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First published online 24 April 2007
doi: 10.1242/jcs.003772

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Phosphoinositide 3-kinase signalling regulates early development and developmental haemopoiesis

Department of Pharmacy and Pharmacology and the Centre for Regenerative Medicine, University of Bath, Claverton Down, Bath, BA2 7AY, UK

View larger version (38K): [in this window] [in a new window]   Fig. 1. Inhibition of PI3Ks impairs EB formation. EBs were generated in the presence of 0, 5 and 10 µM LY294002 (LY) as described in Materials and Methods. (A) Examples of day 5 EBs, original magnification 100x (10x/0.4 objective lens was used). Bars, 200 µm. (B) On day 3 to day 6 samples of EBs were trypsinized and individual cells counted. Data shown are mean (± s.e.m.) from five independent experiments. (C) The total number of EBs formed were counted on day 6. Data shown are mean (± s.e.m.) from four independent experiments. (D) Protein extracts were prepared from ES cells (ES) and EBs on the days indicated and separated by SDS-PAGE. Immunoblots were probed with an antibody detecting phosphorylated serine residues Ser235 and Ser236 of the ribosomal S6 protein (-P-S6). The same immunoblot was stripped and reprobed for SHP-2 to assess equal protein loading. *P<0.05, **P0.01, ***P<0.005.

View larger version (34K): [in this window] [in a new window]   Fig. 2. EBs formed from PDK1-/- ES cells are reduced in size and cellularity. EBs were generated from wild-type parental (WT) and PDK1-/- ES cells. (A) Examples of day 5 EBs, original magnification 100x (10x/0.4 objective lens was used). Bars, 200 µm. (B) On day 3 to day 6, samples of EBs were trypsinised and the cells counted. Data shown are the mean (± s.e.m.) from two to three independent experiments. (C) The mean number of EBs formed, counted on day 6 (± s.e.m.) from three independent experiments. (D) Protein extracts from EBs formed from wild-type (WT) or PDK1-/- (ko) ES cells were prepared on the days indicated, separated by SDS-PAGE and immunoblotted with antibody against phosphorylated S6 (-P-S6). The blot was then stripped and reprobed for GAPDH. *P<0.05, ***P<0.005.

View larger version (42K): [in this window] [in a new window]   Fig. 3. Inhibition of PI3K-dependent signalling does not block haemopoiesis in early development but, rather, enhances it. (A) The number of haemopoietic colonies formed by day 6 EB-derived cells differentiated in the absence (EB 0LY/HCA 0LY) or presence of 5 µM LY294002 (EB 5LY/HCA 0LY). Colonies were defined as follows: primitive erythroid (Ery/P), definitive erythroid (Ery/D), myeloid (including macrophage and granulocyte/macrophage colonies), mixed lineage (including erythroid-macrophage colonies and multi-lineage colonies), and secondary EBs (EB). Data are the mean of quadruple counts (± s.e.m.) from two independent experiments, *P<0.05; **P0.01. (B) Images of day 6 erythroid colonies (left panels) and day 12 haemopoietic colonies (right panels) formed by day 6 WT or PDK1-/- EB cells [original magnification 100x (left panels) or 20x (right panels)]. Bars, 400 µm. Arrow indicates erythroid colony. (C) FACS analysis of Kit (c-kit) and Flk-1 expression of cells derived from EBs formed in the absence (0 µM LY) or presence (5 µM LY) of 5 µM LY294002 or formed from PDK1-/- ES cells. The days of differentiation are indicated. Numbers in each quadrant represent the percent of the total live cells in each sample. Similar trends were observed in three independent experiments.

View larger version (78K): [in this window] [in a new window]   Fig. 4. Inhibition of PI3K-dependent signalling enhances expression of haemopoietic marker genes. (A) RT-PCR analysis of wild-type EBs formed in the absence (0 LY) or presence of 5 µM LY294002 (LY). RT-PCR analysis of EBs formed from wild-type or PDK1-/- ES cells. ES, undifferentiated ES cells; numbers indicate the day of EB differentiation. Data are representative of three (A) and two (B) individual experiments. In A the samples for Scl, Gata-1, β-H1 and β-major were all separated on the same gel but, for simplification of presentation, blank lanes were removed between ES and 0LY d3 samples and between 0LY d7 and 5 µM LY d3 samples. (C,D) Quantitative RT-PCR analysis showing relative levels of the indicated RNA normalised to β-actin. (C) The average and s.d. of three to four replicates from the same experimental RNA as in A are shown and are representative of three independent experiments. White bars, RNA from EBs generated in the absence of LY294002; black bars, RNA samples from EBs generated in the presence of 5 µM LY294002. (D) The mean and ±s.e.m. of quadruple samples from two independent experiments are shown (n=8). White bars, RNA from EBs formed from wild type EBs; black bars, RNA samples from EBs formed from PDK1-/- ES cells. *P<0.05, **P<0.01, ***P0.005.

View larger version (44K): [in this window] [in a new window]   Fig. 5. PI3K-dependent signalling is required for formation of blast colonies. (A) EBs were formed in the presence of 0 or 5 µM LY294002 (LY). EB cells were harvested on day 3, 3.75 or 4 as indicated and identical cell numbers (5x104/ml) plated under blast culture conditions. Examples of blasts cultured for 4 days; original magnification 100x (10x/0.4 objective lens was used). Bars, 200 µm. (B) Following 4 days in culture, the blast colonies were counted. Data represent the average of triplicate counts (± s.d.) and are representative of four independent experiments. (C) EBs were formed from either untreated wild-type or PDK1-/- ES cells and cells were harvested following 3.75 days in culture. Identical cell numbers (3x104/ml) were then plated under blast culture conditions. Cells from EBs generated from the wild-type ES cells were plated either in the absence (0 LY) or the presence of 5 µM LY294002. Examples of blasts cultured for 4 days; original magnification 100x (10x/0.4 objective lens was used). Bars, 200 µm. (D) Following 4 days in culture the blast colonies were dissociated and the total number of cells counted. Data represent mean (± s.e.m.) from three individual experiments. **P0.01 (E and F) RT PCR analyses of 2° blasts. Wild-type or PDK1-/- day 3.75 EB-derived cells were plated under blast culture conditions for 2-4 days. Cells from EBs generated from untreated wild-type ES cells were plated in the absence (0 LY) or presence of 5 µM LY294002 (5 µM LY). RNA was isolated from the initial day 3.75 EB cells (EB) and from the resulting blasts (days of differentiation are indicated) and used for RT-PCR analysis of the marker genes depicted. Note that the EB sample for Scl in E appears in the second lane.

View larger version (23K): [in this window] [in a new window]   Fig. 6. PI3K-dependent signalling is required for optimal development of erythroid and myeloid lineages. (A) Day 3.75 EB-derived cells generated in the absence of inhibitor were plated into blast colony culture conditions in the absence (EB 0LY/blast 0LY) or presence (EB 0LY/blast 5LY) of 5 µM LY294002. In addition, cells from day 3.75 EBs formed in the presence of 5 µM LY294002 were plated under blast culture conditions without inhibitor (EB 5LY/blast 0 LY). After 4 days, blast-colony-derived cells were harvested and identical numbers of cells for each condition plated into HCAs (in the absence of LY294002). Numbers of each type of haemopoietic colony generated are shown. Data represent the average of triplicate counts (± s.d.) and are representative of three individual experiments. ***P<0.005 (B) Day 6 EB-derived cells, differentiated in the absence of LY294002, were plated into HCAs in the absence (EB 0LY/HCA 0LY) or presence of 5 µM LY294002 (EB 0LY/HCA 5LY). Data represents the mean (±s.e.m.) from two independent experiments (n=3-5). Examples showing the size differential of Ery/D colonies that developed in the absence or presence of LY294002 are shown in the inserts; original magnification 100x (10x/0.4 objective lens was used). Bars, 200 µm. These results are representative of five individual experiments, *P<0.05; ***P<0.005. (C) Mouse bone marrow cells were plated into HCAs in the absence (0 LY) or presence of 5 or 10 µM LY294002 (5 µM LY or 10 µM LY, respectively). Data represent the mean (± s.e.m.) of three independent experiments. Examples of overall colony formation are depicted above the appropriate bar graph; original magnification 20x (10x/0.4 objective lens). Bars, 1 mm. In addition, colony morphologies are shown in the inserts; original magnification 100x (10x/0.4 objective lens). Bars, 200 µm. ***P<0.005 for GM colonies compared with 0 LY.

View larger version (11K): [in this window] [in a new window]   Fig. 7. PI3K-dependent signalling is required at multiple stages of developmental haemopoiesis. Our studies have demonstrated that PI3K signalling is required for proliferation of cells within the developing EB at the early stages of development. However, inhibition of PI3K/PDK1-mediated signals does not block differentiation towards the BL-CFC, as indicated by Flk-1 and brachurury expression and blast colony formation. A crucial requirement for PI3Ks and PDK1 was defined during the expansion of the BL-CFC to form blast colonies and this appears to be mainly at the level of proliferation. Inhibition of PI3Ks only during the HCAs indicates PI3Ks are required for optimal generation of myeloid lineages and optimal proliferation of erythroid cells.

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