Phosphoinositide 3-kinase signalling regulates early development and developmental haemopoiesis

Abstract

Phosphoinositide 3-kinase (PI3K)-dependent signalling regulates a wide variety of cellular functions including proliferation and differentiation. Disruption of class I(A) PI3K isoforms has implicated PI3K-mediated signalling in development of the early embryo and lymphohaemopoietic system. We have used embryonic stem (ES) cells as an in vitro model to study the involvement of PI3K-dependent signalling during early development and haemopoiesis. Both pharmacological inhibition and genetic manipulation of PI3K-dependent signalling demonstrate that PI3K-mediated signals, most likely via 3-phosphoinositide-dependent protein kinase 1 (PDK1), are required for proliferation of cells within developing embryoid bodies (EBs). Surprisingly, the haemopoietic potential of EB-derived cells was not blocked upon PI3K inhibition but rather enhanced, correlating with modest increases in expression of haemopoietic marker genes. By contrast, PDK1-deficient EB-derived progeny failed to generate terminally differentiated haemopoietic lineages. This deficiency appeared to be due to a requirement for PI3K signalling during the proliferative phase of blast-colony-forming cell (BL-CFC) expansion, rather than as a result of effects on differentiation per se. We also demonstrate that PI3K-dependent signalling is required for optimal generation of erythroid and myeloid progenitors and their differentiation into mature haemopoietic colony types. These data demonstrate that PI3K-dependent signals play important roles at different stages of haemopoietic development.

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 5 independent experiments. (C) The total number of EBs formed were counted on day 6. Data shown are mean (± s.e.m) from 4 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 serines 235/236 of the ribosomal S6 protein (α-P-S6). The same immunoblot was stripped and reprobed for SHP-2 to assess equal protein loading. *, p<0.05; **, p≤0.01; ***, p<0.005.

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 trypsinized and the cells counted. Data shown are the mean (± s.e.m) from 2-3 independent experiments. (C) The mean number of EBs formed, counted on day 6 (± s.e.m) from 3 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 the α-phospho-S6 antibody (α-P-S6). The blot was then stripped and reprobed for GAPDH. *, p<0.05; ***, p<0.005.

Fig. 3

Inhibition of PI3K-dependent signalling does not block early developmental haemopoiesis, 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; **, p≤0.01. (B) Images of day 6 erythroid colonies (left panels) and day 12 haemopoietic colonies (right panels) formed from 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 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 3 independent experiments.

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) or (B) formed from PDK1^-/- ES cells. ES represents undifferentiated ES cells with numbers indicating the day of EB differentiation. These data are representative of 3 (A) and 2 (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 and D) Quantitative real-time PCR analysis was performed and relative levels of the indicated RNA normalised to β-actin are shown. (C) The average and s.d. of 3-4 replicates from the same experimental RNA as in A are shown and are representative of 3 independent experiments. Open boxes represent RNA from EBs generated in the absence of LY294002 and filled boxes indicate RNA samples from EBs generated in the presence of 5 μM LY294002. (D) The mean and s.e.m. of quadruple samples from 2 independent experiments are shown (n=8); open boxes represent RNA from EBs formed from wild type EBs and filled boxes indicate RNA samples from EBs formed from PDK1^-/- ES cells. *, p<0.05; **, p<0.01; ***, p≤0.005.

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). The EB cells were harvested on day 3, 3.75 and 4 as indicated and identical cell numbers (5×10⁴/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 number of blast colonies were counted. Data represent the average of triplicate counts (± s.d.) and are representative of 4 independent experiments. (C) EBs were formed from either untreated wild type or PDK1^-/- ES cells and the cells harvested following 3.75 days in culture. Identical cell numbers (3×10⁴/ml) were then plated in 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 represents mean (± s.e.m). from 3 individual experiments. **, p≤0.01 (E and F) RT PCR analyses of 2° blasts. Wild type or PDK1^-/- day 3.75 EB-derived cells were plated into 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 (day 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.

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 d3.75 EBs formed in the presence of 5μM LY294002 were plated into blast cultures 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). The numbers of each type of haemopoietic colony generated are shown. Data represent the average of triplicate counts (± s.d.) and are representative of 3 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 2 independent experiments (n=3-5). Examples showing the size differential of Ery/D colonies 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 5 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 and 10 μM LY294002. Data represents the mean (± s.e.m) of 3 independent experiments. Examples showing 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.

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 critical 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.