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.2016 Sep 22;128(12):1567-77.
doi: 10.1182/blood-2016-03-708164. Epub 2016 Jul 15.

Developing HSCs become Notch independent by the end of maturation in the AGM region

Affiliations

Developing HSCs become Notch independent by the end of maturation in the AGM region

Céline Souilhol et al. Blood..

Abstract

The first definitive hematopoietic stem cells (dHSCs) in the mouse emerge in the dorsal aorta of the embryonic day (E) 10.5 to 11 aorta-gonad-mesonephros (AGM) region. Notch signaling is essential for early HSC development but is dispensable for the maintenance of adult bone marrow HSCs. How Notch signaling regulates HSC formation in the embryo is poorly understood. We demonstrate here that Notch signaling is active in E10.5 HSC precursors and involves both Notch1 and Notch2 receptors, but is gradually downregulated while they progress toward dHSCs at E11.5. This downregulation is accompanied by gradual functional loss of Notch dependency. Thus, as early as at final steps in the AGM region, HSCs begin acquiring the Notch independency characteristic of adult bone marrow HSCs as part of the maturation program. Our data indicate that fine stage-dependent tuning of Notch signaling may be required for the generation of definitive HSCs from pluripotent cells.

© 2016 by The American Society of Hematology.

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Figures

Figure 1
Figure 1
Expression of Notch receptors in HSC lineage in AGM region. (A) Expression levels of Notch receptors assessed by qRT-PCR in endothelial cells (VC+CD45−CD43−), pre-HSC type I (VC+CD45−CD43+) and pre-HSC type II (VC+CD45+) sorted from the E11.5 AGM region (n = 3). Data are mean ± standard error of the mean (s.e.m.). *P < .05, **P < .01. (B) Expression of Notch1 and Notch2 in HSC lineage. FACS analysis representing Notch1 or Notch2 presence at the surface of endothelial cells (VC+CD45−CD41−), pre-HSC type I (VC+CD45−CD41+) and pre-HSCs type II (VC+CD45+) in the E11.5 AGM region (n = 3). The graphs on the right indicate mean fluorescence intensity (MFI) ratios between Notch1 or Notch2 and their respective FMO controls during the endothelial-to-pre-HSC transition in 3 independent experiments. Data are mean ± s.e.m. *P < .05. (C) All functional pre-HSCs express Notch1. E11.5 AGM cells were sorted based on Notch1 expression and 2 populations (Notch1+ and Notch1−) were coaggregated with OP9 cells and cultured for 5 days before transplantation into irradiated mice in order to functionally assess the presence of pre-HSCs (0.5 ee per recipient). n = 2; *P < .05, Mann-WhitneyU test. (D) Notch2 is expressed in functional pre-HSC type II, but not in pre-HSC type I. E11.5 VC+CD45− cells (type I) and VC+CD45+ (type II) were sorted based on Notch2 expression level and coaggregated with OP9. After 5 days of culture, they were injected into irradiated recipients (pre-HSC type I: 1 ee per recipient; pre-HSC type II: 0.1 ee per recipient); n = 2; ***P < .005, Mann-WhitneyU test. ns, nonsignificant,t test.
Figure 2
Figure 2
Notch activity decreases during HSC maturation. (A) Expression of Hes1-GFP in endothelial cells (VC+CD45−CD43−; gate R1), pre-HSC type I (VC+CD45−CD43+; gate R2), and pre-HSC type II (VC+CD45+CD43+Sca1+; gate R3) defined by flow cytometry in the E11.5 Hes1-GFP+ AGM region. FMO GFP control (FMO control) was performed with wild-type cells. (B) Pre-HSCs type I are mainly Hes1-GFP+, whereas pre-HSCs type II reside in both the GFP− and GFP+ fraction. VC+CD45− (Pre-HSC type I) and VC+CD45+ (pre-HSC type II) cells were sorted from E10.5 and E11.5 AGM based on Hes1-GFP expression, coaggregated with OP9 cells and transplanted after culture (1 ee per recipient); n = 3. Levels of engraftment are plotted and number of repopulated vs total number of transplanted mice are shown in brackets (***P < .005, Mann-WhitneyU test). (C) AGM dHSCs reside in both Hes1-GFP+ and Hes1-GFP− populations. CD45+ cells were sorted from E12.5 AGM based on Hes1-GFP expression and directly transplanted into irradiated mice (4 ee per recipient); n = 2. (D) Expression of Hes1-GFP in E14.5 fetal liver dHSCs, phenotypically defined by Lin−cKit+Sca1+CD48−CD150+. Gray histogram: Hes1-GFP− control. (E) Fetal liver (FL) HSCs reside in the GFP−/low fraction. LSK populations were sorted based on Hes1-GFP expression from E13.5 and E14.5 fetal liver and directly transplanted into irradiated mice; (0.2 ee per recipient); n = 2. LSK, Lin−Sca+cKit+; LT-HSC, long-term HSC; SSC-A, side scatter.
Figure 3
Figure 3
Forced Notch activity blocks pre-HSC type I maturation. (A-B) Forced activation of Notch activity elevates Hes1-GFP expression in pre-HSCs type I and type II. Hes1-GFP+ pre-HSCs type I (lin−VC+CD45−CD43+cKit+) and type II (lin−VC+CD45+Sca1+cKit+) were sorted from E11.5 AGM and coaggregated either with OP9-WT (left column) or OP9-expressing DLL1 upon doxycycline addition (right columns). After 1 day of culture, VC+CD45+ cells (dot plots) derived from pre-HSCs type I and type II were analyzed for Hes1-GFP expression (black histograms) and compared with Hes1-GFP− control cells (gray histograms). The data are representative of 2 independent experiments. (C) Forced Notch activity prevents pre-HSC type I maturation. Hes1-GFP+lin−VC+CD45−CD43+cKit+ pre-HSCs type I and Hes1-GFP+lin−VC+CD45+Sca1+cKit+ pre-HSCs type II were sorted from E11.5 AGM regions and reaggregated with OP9-Dll1. The coaggregates were cultured with cytokines with or without doxycycline for 5 days before transplantation (0.1 ee per recipient); n = 2; **P < .01, Mann-Whitney test.
Figure 4
Figure 4
Notch signaling is required for pre-HSC development. (A) DAPT treatment completely prevents HSC development in E10.5 AGM, but has a milder effect at E11.5. E10.5 (n = 2) and E11.5 (n = 2) explants were cultured for 5 days without cytokines in the presence of dimethyl sulfoxide (DMSO) or 50 μM DAPT. At the end of the culture, the explants were dissociated and injected into irradiated mice (0.3 ee per mouse); **P < .01, Mann-Whitney test. (B) DAPT treatment disrupts pre-HSC type II maturation. E11.5-sorted VC+CD45+ cells (pre-HSC type II) were coaggregated with OP9. After 5 days of culture with cytokines in presence of DMSO or 50 μM DAPT, the reaggregates were dissociated and injected into irradiated mice (0.2 ee per mouse); n = 3, **P < .01, Mann-Whitney test. (C) DAPT treatment does not affect CFU-C development. E10.5 explants were cultured for 5 days without cytokines in presence of DMSO or 50 μM DAPT. After culture, the development of hematopoietic progenitors (CFU-C) was assessed by performing a colony-forming assay. Bars represent the average number of CFU-Cs per ee and standard errors (n = 3). (D) DAPT does not affect pre-HSC type II maturation into HSCs when added after 24 hours of culture. E11.5-sorted Hes1-GFP+ pre-HSCs type II were cultured for 24 hours prior to addition of DMSO or DAPT. After a subsequent 4 days in culture, the cells were injected into irradiated mice (0.2 ee per mouse); n = 2. (E) Conditional deletion of RBP-Jκ in the E10.5 AGM region. E10.5 RBP-JCKO, RBP-JHet, and wild-type (wt) AGM cells were dissociated, treated individually with 4-OHT for 2 hours, and then cultured as reaggregates without OP9 and without cytokines for 5 days before transplantation. The red triangles represent the recipients whose bone marrow contained RBP-JΔ/Δ dHSCs and showed T-cell phenotype; the blue triangle represents the mouse repopulated with RBP-Jflox/Δ dHSCs (normal T-cell development); black triangles represent recipients whose bone marrow was not analyzed further (0.2 ee per mouse). (F) Numbers of CFU-Cs per ee in E10.5 RBP-JCKO (CKO), RBP-JHet (Het), and WT AGM after culture (4, 3, and 4 embryos, respectively; standard errors are shown). (G) Presence of RBP-Jκ protein after induction of the deletion. E10.5 RBP-JCKO AGM was dissociated and divided into 2 parts, 1 was treated with 4-OHT and the other with methanol (4-OHT vehicle) for 2 hours at 37°C. Twenty-four hours after induction, the presence of RBP-Jκ protein was analyzed by flow cytometry. The dot plots are representative of 4 different AGMs, gated on live cells (EMA−). BFU-E, blood-forming unit-erythrocyte; GM, granulocyte-macrophage; M, macrophage; Mast, mast colonies.
Figure 5
Figure 5
Blocking Notch1 or Notch2 negatively affects HSC development. (A) Both Notch1 and Notch2 are involved in HSC development in the E10.5 AGM region. E10.5 AGM were cultured as reaggregates for 5 days without cytokines in the presence of anti-NRR1 (10 μg/mL) or anti-NRR2 (10 μg/mL) antibodies before transplantation into irradiated mice (0.3 ee per recipient); n = 2, *P < .05, Mann-Whitney test. (B) Blocking Notch1 or Notch2 affects pre-HSC type II maturation. E11.5 pre-HSCs type II (VC+CD45+) were coaggregated with OP9 cells with cytokines and cultured for 5 days with anti-NRR1 (10 μg/mL) or anti-NRR2 (10 μg/mL) antibodies before transplantation (0.1 ee per recipient); n = 2, *P < .05, Mann-Whitney test. (C) Blocking Notch1 or Notch2 does not disrupt CFU-C development. The development of hematopoietic progenitors in the presence of Notch-blocking antibodies after 5 days in culture, in E10.5 AGM reaggregates was assessed by CFU-C assay. The number of hematopoietic progenitors per 1 ee and standard errors are shown (n = 2). GEMM, granulocyte-erythrocyte-macrophage-megakaryocyte.
Figure 6
Figure 6
Model. In the E10.5 AGM region, Hes1-GFP is expressed in all functional pre-HSCs type I, demonstrating that the Notch pathway is activated in these cells. Notch1 is the main receptor at this stage; later on, Notch2 is upregulated during the pre-HSC type I to pre-HSC type II transition. Although both Notch1 and Notch2 are expressed in maturing pre-HSCs and dHSCs, Notch activity decreases because some pre-HSCs and dHSCs at E11.5 and E12.5, respectively, become Hes1-GFP−. Functional analysis showed that Notch activity is essential during the first steps of pre-HSC development. However, the decrease of Notch activity is accompanied by a progressive loss of Notch dependency, as some E11.5 pre-HSCs can complete development in the absence of Notch. In the fetal liver, HSCs either fully lack Notch activity or exhibit it at a low level, despite the presence of both Notch1, and Notch2 at their surface.
See this image and copyright information in PMC

Comment in

  • Turning it down a Notch.
    de Bruijn M.de Bruijn M.Blood. 2016 Sep 22;128(12):1541-2. doi: 10.1182/blood-2016-08-729483.Blood. 2016.PMID:27658698No abstract available.

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