Notch signaling specifies megakaryocyte development from hematopoietic stem cells - PubMed (original) (raw)

Notch signaling specifies megakaryocyte development from hematopoietic stem cells

Thomas Mercher et al. Cell Stem Cell. 2008.

Abstract

In the hematopoietic system, Notch signaling specifies T cell lineage fate, in part through negative regulation of B cell and myeloid lineage development. However, we unexpectedly observed the development of megakaryocytes when using heterotypic cocultures of hematopoietic stem cells with OP9 cells expressing Delta-like1, but not with parental OP9 cells. This effect was abrogated by inhibition of Notch signaling either with gamma-secretase inhibitors or by expression of the dominant-negative Mastermind-like1. The importance of Notch signaling for megakaryopoietic development in vivo was confirmed by using mutant alleles that either activate or inhibit Notch signaling. These findings indicate that Notch is a positive regulator of megakaryopoiesis and plays a more complex role in cell-fate decisions among myeloid progenitors than previously appreciated.

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Figures

Figure 1

Figure 1. The DL1/Notch Axis Induces Megakaryocyte Differentiation In Vitro

(A) Flow-sorted LSK cells were plated on OP9-GFP or OP9-DL1 stroma. (Left panels) Phase-contrast microscopy of day 8 LSK cells/OP9-GFP or OP9-DL1 stroma cocultures. Arrowheads indicate megakaryocytes observed in OP9-DL1 cocultures. Original magnification is ×400. (Middle and right panels) Acetylcholinestesterase (AchE) staining of cytospun cells from cocultures. Original magnification was ×100 and ×600, respectively. Brown coloring indicates positivity for AchE. (B) Flow cytometric analysis of CD45+ cells derived from day 8 LSK/OP9-GFP or OP9-DL1 cocultures. (C) Histogram representation of flow cytometric results presented in (B). Mean ± SEM of five independent flow cytometric analyses after 8 days of coculture is shown. (D) γ-secretase inhibition abrogates biological and molecular effects of DL1 stimulation. LSK cells cultured on OP9-DL1 stroma in the presence of DMSO (control) or Compound E (CompE, 1 µM) were analyzed for megakaryocytic differentiation.

Figure 2

Figure 2. RBPJ/ICN/MAML Complex Mediates Megakaryocyte Development

(A and B) LSK cells were transduced with retroviruses encoding either dnMAML1, ICN1, ICN4, HES1, HES5, or MIG-empty vector control and subsequently plated on OP9-GFP or OP9-DL1 stroma. (C) LSK cells were transduced as in (A) and (B) and plated in presence of 1 µM Compound E or mock (DMSO) control. FACS analyses of CD45+GFP+ cells were performed after 8 days of coculture.

Figure 3

Figure 3. Notch Activates a Megakaryocyte-Specific Transcriptional Program in LSK Cells

(A) RNA from LSK cells cocultured with OP9-GFP or OP9-DL1 in the presence or absence of Compound E (1 µM) for 5 days was used to perform analysis of Hes-1, Gata-1, Fli-1, and PU.1 expression normalized to Gapdh. Mean ± SEM of duplicate experiments is represented. (B) Flow-sorted LSK cells were cultured on stroma (OP9-GFP, OP9-DL1, or OP9-DL1 supplemented with 1 µM Compound E). After 3 days of cocultures, RNA from nonadherent cells was extracted, amplified, labeled, and hybridized on mouse 430.2E Affymetrix chips. Expression data were analyzed for a list of genes positively involved in megakaryopoiesis by using GSEA. Enrichment plot showing upregulation of megakaryocyte-specific genes in OP9-DL1 versus OP9-GFP and OP9-DL1+Compound E. P value and FDR are indicated. (C) Heat map representation of the expression of the top 50 megakaryocyte leading edge genes enriched in OP9-DL1 cultures compared to OP9 and OP9-DL1 supplemented with inhibitor cultures. (D) Venn diagram representation of megakaryocytic genes induced by Notch pathway activation in LSK cells.

Figure 4

Figure 4. Notch Specifies Megakaryocyte Fate at Several Levels of Hematopoietic Differentiation

(A) Limiting dilution assay with LSK cells from wild-type murine bone marrow directly sorted into individual OP9-GFP or OP9-DL1-coated 96-well plates with 1, 2, 5, or 20 cells per well and cultured for 8 days. Cocultures were analyzed under a microscope: wells with visible hematopoietic cells were scored and megakaryocytes were counted. Frequency of megakaryocyte-containing wells is represented, and trend lines were used to estimate the frequency of megakaryocyteforming cells in the LSK population. (B) Flow-sorted Lin−Sca1+Kit+CD34−Flt3− (LT-HSC), Lin−Sca1+Kit+CD34+Flt3− (ST-HSC), and Lin−Sca1+Kit+CD34+Flt3+ (LMPP) cells were plated directly onto OP9-GFP or OP9-DL1 stroma. Flow cytometric analysis was performed after 7 days of coculture. (C and D) RNA from flow-sorted CMP, MEP, and GMP were extracted, amplified, and used for analysis of quantitative expression of Notch receptors (C) and the Notch targets Hes-1, Hes-5, and Hey-1 (D). Results were normalized to GAPDH expression and to the MEP value. Notch3 expression was not detected in any sample. Error bars represent SEM. (E) Flow-sorted CMP were cultured on OP9-GFP or OP9-DL1 for 5 days before analysis by flow cytometry. (F) Flow-sorted MEP were cultured on OP9-GFP or OP9-DL1 for 3 days before analysis by flow cytometry.

Figure 5

Figure 5. DnMAML1 Inhibits Megakaryocyte Development In Vivo

(A) Wild-type bone marrow cells were infected with dnMAML1-encoding or MIG control retroviruses and injected into lethally irradiated syngeneic recipients. Analysis was performed after 3–6 weeks. Flow cytometric analysis of myeloid progenitors within the Lineage−cKit+Sca1− population. Analysis was gated on GFP+ cells. A representative of five independent animals is shown for each group. Mean ± SEM for a total of five independent animals is represented below as histograms. (B) Analysis of the megakaryocyte progenitors c-Kit+CD41+ population (gated on Lineage− GFP+cells) in the bone marrow of MIG versus dnMAML1 recipient animals. (C) FACS analyses of GFP+ dnMAML1 recipient bone marrow cells indicate impaired megakaryocyte development compared to MIG control recipients. (D) Immunohistochemical (IHC) analysis of consecutives bone marrow sections shows reduced staining for the megakaryocyte-specific vWF and mostly GFP− megakaryocytes in dnMAML1 recipients, compared to MIG controls. For both vWF and GFP immunostainings, positive cells show a dark brown color. Black and white arrowheads indicate GFP+ and GFP− megakaryocytes, respectively. Original magnifications are ×100 (vWF) and 31000 (GFP). (E) Fifty megakaryocytes were counted on bone marrow sections stained for GFP, and the percentages of GFP+ megakaryocytes are shown (white histograms). The percentages of GFP+ total bone marrow (BM) cells were assessed in the same recipients by flow cytometry and are also shown (black histograms). Histograms represent mean ± SEM of three independent animals for each group. (F) Ploidy analysis was performed by flow cytometry using propidium iodide and gating on GFP+CD41+ megakaryocytes. Median ploidy is indicated above the histogram (value for diploid state = 2).

Figure 6

Figure 6. DnMAML1 Conditional Knockin Mice Have Impaired Megakaryopoiesis

(A) DnMAML1 cKI-Mx1Cre double transgenic animals were induced with poly(I:C) at 6 weeks of age and analyzed 2–3 weeks later. A representative flow cytometric analysis of myeloid progenitors within the Lineage−cKit+Sca1− population is shown. Mean ± SEM of three independent analyses gated on GFP+ cells is shown below as histograms. (B) Flow-sorted CMPs were plated in methylcellulose cultures supplemented with IL3, IL6, SCF, EPO, and TPO, and colonies were counted after 7 days. GM, granulocyte-macrophage colony; GEMM, granulocyte-macrophage-erythroid-megakaryocyte colony; EMk, erythroid-megakaryocyte colony; E, erythroid colony; Mk, megakaryocyte colony; cKI, conditional knockin. Mean ± SEM (n = 3) are shown.

Figure 7

Figure 7. ICN4 Supports Megakaryocyte Development In Vivo

(A) Rag1−/− bone marrow cells were infected with ICN4-encoding or MIG control retroviruses and injected into wild-type lethally irradiated C57BL/6 recipients. The MEP population was analyzed by flow cytometry. Representative analysis of myeloid progenitor populations in ICN4 recipients compared to controls (GFP+ or GFP− gated population is indicated on the left side). (B) Absolute numbers of MEP are indicated as mean ± SEM of three independent analyses. (C) Megakaryocyte colony-forming unit (CFU-MK) potential from total bone marrow. Mean ± SEM of quadruplicate experiments is represented. (D) Consecutive bone marrow sections from ICN4 and MIG recipients were stained as in Figure 5D. Arrowheads indicate GFP+ megakaryocytes. Original magnification in the upper left four panels is ×100 and in the upper right panel is ×1000. (E) The number of GFP+ megakaryocytes and bone marrow (BM) cells were assessed as in Figure 5E. Histograms represent mean ± SEM of three independent animals in each group. (F) Flow cytometric analysis of megakaryocyte ploidy gated on GFP+CD41+ cells. Median ploidy is indicated in the histograms (value for diploid state = 2). (G) Flow-sorted MEPs from the bone marrow of wild-type C57BL/6 animals were retrovirally transduced with empty control (MIG), ICN1, or ICN4 retroviruses and injected into lethally irradiated recipients with 2 × 105 helper bone marrow cells. Bone marrow cells from recipients were analyzed 12 days posttransplant and analysis was gated on GFP+ cells.

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