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First published online 17 September 2008
doi: 10.1242/dev.025916

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Numb mediates the interaction between Wnt and Notch to modulate primitive erythropoietic specification from the hemangioblast

¹ Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
² Stem Cell and Developmental Biology Department, Genome Institute of Singapore, 138672 Singapore.
³ McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario M5G 1L7, Canada.

View larger version (52K): [in this window] [in a new window]   Fig. 1. Gene expression analysis of developing blast colonies. Flk1+ cells isolated from Bry-GFP ES cell-derived day 3.25 EBs were cultured in serum-containing hemangioblast methylcellulose media and the developing blast colonies assayed at the indicated time intervals. (A) Images showing the morphology of representative developing blast colonies over a 72-hour culture period. Scale bars: 10 µm. (B) Expression profiles of individual developing blast colonies isolated at the indicated times. Each lane represents an individual colony. Pos, positive control. (C) Hematopoietic progenitor potential of different aged blast colonies. Seven-hundred colonies of each time point were collected, dissociated and the cells plated in methylcellulose media supplemented with hematopoietic cytokines. Primitive erythroid (Ep), macrophage (Mac) and bipotential macrophage/erythroid (Emac) colonies were scored after 4-5 days of culture, while definitive erythroid (Ed) and multipotential myeloid/erythroid (Mix) colonies were scored at day 9 of culture. (D) Expression of Notch and Wnt signaling components in developing blast colonies revealed by quantitative PCR. 3' cDNA samples of day 3.25 Flk1+ cells (0 hours) and 3' cDNAs of seven individual blast colonies used in B were pooled and analyzed at each time point (12-48 hours). Average expression normalized to Actb is shown. Error bars represent the standard errors of mean from three independent experiments.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Immunostaining of Numb, cleaved Notch1 intracellular domain and active β-catenin in developing blast colonies. Immunostaining showing (A) Numb and activated β-catenin (β-cat), (B) cleaved Notch1 intracellular domain (NIC) and activated β-catenin (β-cat) in 12 hour, 24 hour and 48 hour blast colonies. Merged images show the overlay of three channels (DAPI, Cy2 and Cy3). Light transmission images (DIC) display the morphology of the cells. Arrows indicate the nuclear presence of activated β-catenin. Scale bars: 10 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Wnt and Notch signaling affect blast colony formation from the hemangioblast. (A) Role of Wnt signaling in blast colony development. Flk1+ cells (4x104) isolated from the day 2.75 EBs generated from either sβ-cat, Numb or Notch1-IC ES cells were cultured in M10 serum-free hemangioblast media in the presence or absence of doxycyclin (Dox; 2 µg/ml) and DKK1(DKK; 300 ng/ml) as indicated. Dox was added at 0 hours of culture, whereas DKK1 was added at 6 hours of culture. Colonies were washed with media at 24 hours to deplete DKK1 and Dox, and replated back into fresh M10. Blast and core colonies were scored after 4 days of culture. (B) Effect of DKK1 treatment on blast and core colony development. (C) Hematopoietic potential of DKK1-treated colonies. Sixteen-hundred colonies of each group were picked at day 3 of culture, pooled, dissociated and replated into hematopoietic methylcellulose cultures. Ep, primitive erythroid colonies; Def, combined macrophage, bipotential macrophage/erythroid, definitive erythroid and multipotential myeloid/erythroid colonies. (D) Role of Numb in blast colony development. gSI, -secretase inhibitor (2.5 µM). (E) Role of Notch signaling in blast colony development. (A-E) Error bars represent standard deviations of the mean of number of colonies from n independent experiments (A, n=3; B, n=3; C, n=3; D, n=4; E, n=4; **P<0.01; ***P<0.001)

View larger version (26K): [in this window] [in a new window]   Fig. 4. Wnt and Notch signaling regulate primitive erythroid development from Flk1+ cells. Flk1+ cells isolated from day 2.75 EBs generated from the indicated ES cell lines were cultured as aggregates in serum-free media containing VEGF (10 ng/ml) and either DKK1 (300 ng/ml), Dox (2 µg/ml), -secretase inhibitor (2.5 µM) or DMSO (2.5 µl/ml), as specified. (A) Expression of Axin2 and Wnt3 in aggregates at different times. Average expression normalized to Actb is shown. (B) Transient requirement for Wnt signaling in primitive erythroid specification of Flk1+ cells. Ep, primitive erythroid colonies; Def, combined macrophage, bipotential macrophage/erythroid, definitive erythroid and multipotential myeloid/erythroid colonies. Total colony numbers are shown. (C) Effect of Numb expression or addition of gSI on primitive erythroid development from Flk1+ cells. -Dox: doxycyclin-untreated group; +Dox: doxycyclin was added for the first 24 hours, and then washed away. (D) Effect of Notch expression on primitive erythroid development from Flk1+ cells. Dox was added as indicated above in C. (B-D) Error bars represent standard deviation of the mean number of colonies from n independent experiments (B, n=3; C, for the first three groups n=6, and last two groups n=3; D, n=6;**P-value<0.01; ***P-value<0.001).

View larger version (36K): [in this window] [in a new window]   Fig. 5. Interaction of Wnt and Notch signaling in modulating primitive erythropoiesis from Flk1+ cells. Flk1 cells isolated from day 2.75 EBs generated from the indicated ES cell lines were cultured as aggregates in serum free media containing hVEGF (10 ng/ml) and either Wnt3a (100 ng/ml), DKK1 (300 ng/ml), Dox (2 µg/ml), -secretase inhibitor (gSI; 2.5 µM) or DMSO (2.5 µl/ml), as specified. Hematopoietic potential of the aggregates was assayed as described in the Materials and methods. (A) Synergistic effects of Numb and Wnt3a on primitive erythroid development from Flk1+ cells. (B) Synergistic effects of -secretase inhibitor and Wnt3a on primitive erythroid development from Flk1+ cells. (C) Effect of Wnt3a on Notch1-IC-induced suppression of primitive erythroid development. (D) Effect of Numb expression on DKK1-induced block in primitive erythropoiesis. DKK1 was added to the reaggregation media after 6 hours of culture to allow time for Numb protein to accumulate. (A-D) Error bars represent the standard deviation of the mean of the number of colonies from n independent experiments (A, n=6; B, n=4; C, n=5; D, n=3) (**P-value<0.01; ***P-value<0.001). (E) The TOP/FOPflash reporter assay demonstrates interaction between Wnt and Notch pathways. Error bars indicate standard deviation of TOP/FOP values of triplicate electroporations. Numbers above each bar represent actual TOP/FOP values.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Expression analyses of Flk1+ cell-derived populations following induction of Notch1-IC or Numb. Flk1+ cells isolated from day 2.75 EBs generated from either the Numb or Notch1-IC ES cell line were cultured as aggregates with VEGF (10 ng/ml) in the presence or absence of Dox (2 µg/ml). Flk1+ cells prior to culture (0 hours) or aggregates following 12 hours, 24 hours, and 48 hours of culture were harvested for qPCR. Average expression normalized to Actb is shown. Error bars represent the standard errors of mean from three independent experiments. (A) Sfrp1; (B) Sfrp5; (C) Wnt5a; (D) Nlk; (E) Dkk1.

View larger version (115K): [in this window] [in a new window]   Fig. 7. Expression of Numb, Notch1-IC and activated β-catenin in early embryos. Confocal images (maximum projection by z-stacking, XY planes) of whole-mount immunostaining for (A) Numb, (B) cleaved Notch1 intracellular domain (Notch1-IC) and (C) activated β-catenin in different staged mouse embryos and yolk sacs. Yellow arrows show the yolk sac; white arrowheads indicate the posterior primitive streak region in neural plate stage embryos, and white arrows indicate the embryo proper. Broken lines delineate the YS region in somite-stage embryos. Scale bars: 100 µm. Red, Numb, Notch1-IC or activated β-catenin; blue, DAPI

View larger version (11K): [in this window] [in a new window]   Fig. 8. Model of primitive erythroid development. The changes in the signaling pathways during the first 24 hours of blast colony development are shown. Both Wnt and Numb are indicated as active in the isolated Flk1+ population (0 hours) as well as in the 12-hour colonies. The Notch pathway is not active in the starting Flk1 population, but is activated within the first 12 hours of colony growth. The activation of Notch induces different Wnt inhibitors, which in turn inhibits the Wnt pathway by 24 hours. Arrows below the line indicate specification to the primitive erythroid and definitive hematopoietic lineages. The thicker red arrow at 24 hours represents a higher proportion of primitive erythroid progenitors at this stage, whereas the thicker blue arrow at 48 hours depicts an expansion of the definitive lineages.

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