Phosphoinositide 3-kinase (PI3K)-dependent signalling regulates a wide variety of cellular functions including proliferation and differentiation. Disruption of class IA 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.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases, whose products, phosphatidylinositol (3,4)-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3], act as intracellular second messengers (Cantley, 2002; Vanhaesebroeck et al., 2001; Vanhaesebroeck and Waterfield, 1999). Three classes of PI3K have been described based on their structure and in vitro substrate specificity (Vanhaesebroeck and Waterfield, 1999). Members of the class IA family of PI3Ks are composed of a regulatory subunit (p85 or p55) and a p110 catalytic subunit (α, β or δ), and are activated by numerous growth factors and cytokines (Cantley, 2002; Vanhaesebroeck et al., 2001; Vanhaesebroeck and Waterfield, 1999). 3-phosphoinositide-dependent protein kinase 1 (PDK1, also known as PDPK1) is a direct downstream effector of PI3Ks (Vanhaesebroeck and Alessi, 2000) and plays a central role in activating other downstream AGC-family kinases, ultimately leading to the regulation of a wide array of physiological processes, including proliferation, survival and differentiation (Mora et al., 2004). Gene-targeting studies in mice, in which one or more of the class IA PI3K sub-units has been functionally disrupted, have revealed a requirement for this family of enzymes in regulating developmental events. For example, disruption of both p85α and p85β gene products results in lethality at embryonic day 12.5 (E12.5) (Brachmann et al., 2005), whereas embryos deficient in p110α die between E9.5 and E10.5 due to a proliferative defect (Bi et al., 1999). Importantly, deletion of p110β leads to a very early developmental defect, resulting in embryonic lethality at the pre-implantation blastocyst stage (Bi et al., 2002). Interestingly, class IA catalytic and regulatory subunits are expressed from the single-cell stage of development (Lu et al., 2004; Riley et al., 2005) and PI3K signalling appears to be required for correct development of pre-implantation mouse embryos (Lu et al., 2004; Riley et al., 2005; Riley et al., 2006). The phenotypes of several PI3K knockout mice have highlighted a requirement for PI3K-mediated signalling in development of the haemopoietic system. Disruption of p85α alone has demonstrated a role for PI3Ks in erythropoiesis (Huddleston et al., 2003), whereas p85α-/-/p85β-/+ foetal liver-derived haemopoietic stem cells have recently been reported to have reduced repopulating ability (Haneline et al., 2006). Both p85α and the p110δ subunits have been shown to be involved in B and T cell development (Clayton et al., 2002; Fruman et al., 1999; Okkenhaug et al., 2002; Suzuki et al., 1999). These reports suggest that PI3Ks play an important role during early embryonic development and that PI3Ks may be specifically required during developmental haemopoiesis.

The inaccessibility of early embryonic material and the embryonic lethality of several PI3K gene knockouts have made it difficult to further characterise the requirement for PI3K-mediated signals during early development. Embryonic stem (ES) cells provide a unique in vitro model system in which the contribution of specific signals to early developmental stages can be examined (Doetschman et al., 1985; Keller et al., 1993; Keller, 1995). Extensive genetic analyses have demonstrated that in vitro differentiation of ES cells into embryoid bodies (EBs) recapitulates the events leading to the onset of primitive haemopoiesis in the yolk sac of the normal mouse embryo (Keller, 2005; Keller et al., 1993; Palis et al., 1999). Using ES cell differentiation, a progenitor representing the earliest stage of haemopoietic commitment has been identified (Choi et al., 1998; Nishikawa et al., 1998). This EB-derived progenitor, known as the blast-colony-forming cell (BL-CFC), has both haemopoietic and endothelial potential and can be characterised by co-expression of Flk-1 (also known as Kdr) and the mesodermal gene brachyury (Choi et al., 1998; Faloon et al., 2000; Fehling et al., 2003; Kennedy et al., 1997). The BL-CFC represents the in vitro equivalent of the yolk sac haemangioblast, recently identified in the early mouse embryo (Huber et al., 2004).

To further examine the role of PI3K-dependent signalling pathways during early development and, more specifically, developmental haemopoiesis, we have used ES cells as an in vitro model and employed both pharmacological and genetic approaches to manipulate PI3K activity at different stages of differentiation. LY294002 is a reversible inhibitor (in vitro IC50 1.4 μM) of all classes of PI3K (class IA, IB, II and III), although the activity of PI3K-C2α is refractory to concentrations of LY294002 that inhibit the activity of the other PI3K family members (Knight et al., 2006; Vlahos et al., 1994). LY294002 is stable in solution and can be added or removed from assays at various points, making it a useful tool for deciphering the biological requirement of PI3K signalling at a cellular level. To complement the inhibitor studies and to investigate the role of class IA PI3Ks specifically, we employed ES cells in which PDK1, a crucial downstream effector of class IA PI3Ks, has been disrupted. Using these complementary approaches, we demonstrate that the major role of PI3Ks during the earliest stages of ES cell differentiation is to regulate proliferation. At later stages of differentiation we demonstrate that PI3K-dependent signalling is required for expansion of BL-CFC and haemopoietic precursors. Thus, PI3Ks play multiple roles at defined times during early development and differentiation of the primitive haemopoietic system.

PI3K-dependent signalling is required for optimal EB development

Initially, we examined whether inhibition of PI3K signalling affects the ability of ES cells to develop into EBs. ES cells were differentiated into EBs in the absence or presence of 5 or 10 μM of the PI3K inhibitor LY294002 (Vlahos et al., 1994). As seen in Fig. 1A, the size of EBs decreased upon inhibition of PI3K signalling, correlating with a two- to threefold reduction in cellularity in the presence of 5 μM LY294002 (see Fig. 1B), whereas 10 μM LY294002 almost completely inhibited EB development. Importantly, despite the size reduction, inhibition of PI3Ks with 5 μM LY294002 did not affect the plating efficiency of the ES cells, the same number of EBs being generated irrespective of the presence of the PI3K inhibitor (Fig. 1C). In addition, the decrease in cellularity did not appear to be a result of increased apoptosis, which was not significantly altered upon inhibition of PI3Ks (data not shown). As an indicator of PI3K activity we measured the status of S6 phosphorylation at serine residues Ser235 and Ser236 by immunoblotting (Fig. 1D). S6 phosphorylation was significantly reduced in EBs treated with 5 μM LY294002 and almost completely inhibited with 10 μM LY294002.

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 100× (10×/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 (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. 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 100× (10×/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 (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.

LY294002 inhibits all classes of PI3K, with the exception of PI3K-C2α (Knight et al., 2006; Vlahos et al., 1994) and we wanted to investigate more specifically the role of class IA PI3K. However, the complexity of this family (see Introduction) precluded a genetic knock-out approach of all isoforms within the same ES cell. Therefore, we reasoned that ES cells in which PDK1, a crucial downstream effector of class I PI3K, had been disrupted would provide a robust genetic tool to determine whether the effects we observe with LY294002 are attributable to class I PI3K-dependent signals. ES cells in which both copies of the gene encoding PDK1 (Pdpk1) were disrupted (PDK1-/- ES cells) (Williams et al., 2000) formed EBs that were reduced in size and showed a two- to threefold decrease in cellularity compared with wild-type parental ES cells (Fig. 2A,B), similar to the effects observed with 5 μM LY294002 (Fig. 1). The plating efficiency of the PDK1-/- ES cells was not significantly different from wild-type ES cells (Fig. 2C). S6 phosphorylation could not be detected by immunoblotting extracts from PDK1-/- EBs (Fig. 2D), indicating that PDK1 is required in the pathway leading to phosphorylation of S6, as previously reported (Williams et al., 2000). These data indicate that PI3K signalling pathways, mediated at least in part via PDK1, are required for optimal formation and development of EBs and are consistent with class IA PI3Ks playing a role in early developmental processes.

Inhibition of PI3K-dependent signalling does not block early developmental haemopoiesis but rather enhances the haemopoietic potential of EB-derived cells

Our results demonstrate that PI3K-dependent signalling events are required for optimal development of EBs. We were interested in determining whether haemopoietic differentiation had been affected when PI3K signalling was perturbed during EB formation. To detect the emergence of haemopoietic precursors within the developing EB, EBs were formed in the presence or absence of LY294002 and we examined the ability of day 6 EB-derived cells to generate colonies in haemopoietic-colony-forming assays (HCAs), in the absence of LY294002, using a cocktail of cytokines designed to facilitate maturation of a range of haemopoietic cell types. Somewhat surprisingly, inhibition of PI3Ks during EB development led to an increase in the proportion of EB progeny with haemopoietic potential, evidenced by increases in the number of erythroid, myeloid and mixed colonies present in the HCAs compared with untreated EBs (Fig. 3A). Few secondary EBs developed, indicating efficient differentiation of cells within the EBs. By contrast, when we performed similar experiments with day 6 PDK1-deficient EB-derived cells, only very small erythroid colonies formed (Fig. 3B), whereas by day 12 no colonies were visible. Contrary to inhibition of PI3Ks with LY294002 only during EB formation (Fig. 3A), which measures the effects of PI3K inhibition on progenitor formation within the EB, in this experiment using PDK1-/- ES cells, the class I PI3K pathway would be inhibited throughout the entire experiment, including during formation of colonies in the HCAs. These results suggest that PDK1-dependent signals are required during developmental haemopoiesis, following haemopoietic progenitor formation within the EB. We were interested in further defining the stage at which PI3K signalling was required.

The emergence of different ES-cell-derived haemopoietic populations within the developing EB can be tracked by expression of specific cell surface markers (Kabrun et al., 1997; Mikkola et al., 2003; Nishikawa et al., 1998). Flk-1 is initially expressed on BL-CFCs and represents the onset of embryonic haemopoiesis (Faloon et al., 2000; Fehling et al., 2003; Kabrun et al., 1997), whereas the proto-oncogene product Kit marks cells committed to the haemopoietic lineage and that arise during the early stages of EB development (Fehling et al., 2003; Ling and Neben, 1997; Ogawa et al., 1991). In addition, it has been suggested that Flk-1+-Kit+ double-positive cells represent a population with haemopoietic potential that mark the establishment of haemopoiesis (Lacaud et al., 2004; Willey et al., 2006). The pattern of Flk-1 and Kit surface expression on cells from developing EBs formed in the presence or absence of 5 μM LY294002 or formed from PDK1-/- ES cells was analysed by flow cytometry. In untreated wild-type EBs, little Flk-1 or Kit was expressed on day 3 (results not shown). As shown in Fig. 3C, expression of Flk-1 dramatically increased over the next 24 hours followed by the expression of Kit, which was maximal on day 5 and day 6. Importantly, a Flk-1+-Kit+ double-positive population was observed on day 4 which increased over the next 48 hours. These profiles are similar to those previously described (Kabrun et al., 1997; Lacaud et al., 2004). Inhibition of PI3Ks with 5 μM LY294002 led to no change in overall Kit expression, although a small enhancement in overall Flk-1 expression was observed, particularly on days 5 and 6 (Fig. 3C). Interestingly, the proportion of Flk-1+-Kit+ double-positive cells increased on days 5 and 6 compared with untreated EBs, correlating with the enhanced formation of haemopoietic colonies observed (Fig. 3B). Despite not being able to generate mature haemopoietic colonies, cells from PDK1-/- EBs showed an increase in overall Flk-1 expression, culminating in an enhanced Flk-1+-Kit+ double-positive cell population (Fig. 3C). These findings suggest that inhibition of PI3K signalling does not block formation of early haemopoietic progenitors and may result in an enhanced proportion of EB cells with haemopoietic potential.

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 100× (10×/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 (α-P-S6). The blot was then stripped and reprobed for GAPDH. *P<0.05, ***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 100× (10×/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 (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 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; **P⩽0.01. (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 100× (left panels) or 20× (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.

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; **P⩽0.01. (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 100× (left panels) or 20× (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.

We also examined expression of marker genes to track progression from the mesoderm (brachyury) to the BL-CFC (Flk-1) and on to committed haemopoietic progenitors (Scl and Gata-1) (Chung et al., 2002; D'Souza et al., 2005; Fehling et al., 2003; Fujimoto et al., 2001; Robertson et al., 2000), as well as the globin genes β-H1 (primitive erythroid) and β-major (definitive erythroid). Using semi-quantitative RT-PCR (Fig. 4A and B) inhibition of PI3K-dependent signalling by LY294002 treatment or disruption of PDK1 did not appear to significantly affect either the timing or apparent magnitude of expression of this set of marker genes. However, such semi-quantitative approaches cannot adequately distinguish relatively modest changes in gene expression and in light of the results of our HCAs, we decided to measure expression of these genes by quantitative PCR. Expression of brachyury was reduced in day 3 EBs formed in the presence of LY294002 (Fig. 4C) or derived from PDK1-/- ES cells (Fig. 4D). Despite the increased surface expression of Flk-1 in EBs formed in the presence of LY294002 or from PDK1-/- ES cells, Flk-1 gene expression was reduced in day 4 EBs with a slight enhancement by day 6 in EBs formed in the presence of LY294002 (Fig. 4C and D). In light of the enhanced haemopoietic potential of EB cells derived in the presence of LY294002 observed in our HCAs, we were particularly interested in examining the expression of Scl, Gata-1, β-H1 and β-major. By day 6, all were upregulated two- to threefold in EBs formed in the presence of LY294002, correlating with the enhanced haemopoietic potential of these day 6 EB-derived cells. Taken together, these data indicate that inhibition of PI3K-dependent signalling does not block early stages of haemopoietic differentiation within developing EBs.

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, ***P⩽0.005.

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, ***P⩽0.005.

PI3K-dependent signalling is required for expansion of BL-CFC progeny

Having observed that inhibition of PI3K-dependent signalling within developing EBs does not cause gross changes in the expression of differentiation markers but absence of PDK1 appears to preclude development of terminally differentiated haemopoietic cells, we decided to examine whether PI3K-mediated signalling was involved in the formation or expansion of BL-CFCs, the earliest haemopoietic progenitors. BL-CFCs are transient progenitors that arise in EBs between day 2.5 and day 4 of differentiation, and are able to generate blast colonies with haemopoietic and endothelial potential (Choi et al., 1998; Faloon et al., 2000; Kennedy et al., 1997). BL-CFCs have been characterised by co-expression of Flk-1 and brachyury (Faloon et al., 2000; Fehling et al., 2003; Kabrun et al., 1997). We first determined whether inhibiting PI3Ks affected differentiation towards BL-CFCs within the EBs by assessing the ability of EB-derived cells to form blast colonies. EBs generated in the presence or absence of LY294002 were harvested following 3, 3.75 and 4 days of differentiation, and identical cell numbers were plated under blast-colony-forming conditions with no further inhibitor added. As indicated in Fig. 5A and B, cells from day 3 EBs were unable to form blast colonies but blast colonies were formed by cells from EBs differentiated for 3.75 and 4 days, indicating the emergence of the BL-CFC and correlating with expression of Flk-1 (Fig. 3C) and brachyury (Fig. 4A). Culturing EBs with 5 μM LY294002 had little or no affect on the number of blast colonies formed (Fig. 5B) or their cellularity. These data demonstrate that inhibition of PI3Ks does not block differentiation towards BL-CFCs within EBs. However, when we performed similar experiments with PDK1-/- ES cells no blast colonies formed (Fig. 5C). This was not simply due to a temporal change in developmental potential, because PDK1-/- cells from EBs differentiated for 3-5 days had no blast-colony-forming potential (data not shown). Two possibilities could account for these observations: (1) PDK1-/- ES cells cannot differentiate into BL-CFCs within the EB. However, we feel this is unlikely because these cells express Flk-1 and brachyury in a pattern similar to wild-type parental ES cells (Fig. 3C and Fig. 4B). (2) PI3K signals, mediated at least in part via PDK1, are required for the expansion and/or differentiation of the BL-CFC to form a blast colony. We tested this possibility by assessing whether inhibiting PI3Ks only during blast culture affected blast colony formation. Cells from EBs, formed in the absence of inhibitor for 3.75 days to allow for formation of the BL-CFC, were plated under blast-colony-forming conditions in the presence or absence of 5 μM LY294002. Inhibition of PI3K signalling with 5 μM LY294002 significantly inhibited the ability of BL-CFCs to form archetypal blast colonies - only small clumps of cells were observed (Fig. 5C) - corresponding to a tenfold decrease in cellularity (Fig. 5D). These data indicate that PI3K-dependent signalling is required for BL-CFCs to undergo expansion to form blast colonies.

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 (5×104/ml) plated under blast culture conditions. Examples of blasts cultured for 4 days; original magnification 100× (10×/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 (3×104/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 100× (10×/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. **P⩽0.01 (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.

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 (5×104/ml) plated under blast culture conditions. Examples of blasts cultured for 4 days; original magnification 100× (10×/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 (3×104/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 100× (10×/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. **P⩽0.01 (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.

Blast colony formation has been described as a sequential process involving both proliferation and differentiation events (D'Souza et al., 2005). Within 1-2 days in culture the BL-CFC first proliferates to form a tight core of cells. Haemopoietic cells then develop from the core and after 3-4 days these cells cover the core. Haemopoietic, endothelial and vascular smooth muscle lineages comprise the mature blast colony. Our results could either be due to effects on proliferation - directly affecting growth of the blast colony - or due to effects on differentiation. To distinguish between these two possibilities, we examined the temporal expression of marker genes to track blast colony differentiation. As shown in Fig. 5E, patterns of gene expression were very similar in blast colonies generated when PI3K was inhibited or not, with flow cytometry demonstrating similar cell-surface-expression profiles for Flk-1, Kit and CD41 (data not shown). Intriguingly, despite no outward appearance of PDK1-deficient EB-derived cells being able to form blast colonies, cells recovered from these cultures showed a characteristic downregulation of brachyury as well as expression of genes encoding Scl, Gata-1, β-H1 and β-major (Fig. 5F). These data indicate that there are no dramatic alterations in the blast colony differentiation profiles in the absence of PI3K-dependent signalling; therefore, in light of the decline in size of blast colonies, the most likely explanation of our results is that PI3K-dependent signals are required for proliferation of progenitor cells within the developing blast colony.

PI3K-dependent signalling is required for optimal development of haemopoietic precursors and mature haemopoietic lineages

Our results indicate that PI3K-mediated signals are required for expansion of the BL-CFC to form a blast colony. However, analyses of marker gene expression indicated that haemopoietic differentiation can still occur within the cells which do develop, so we decided to investigate whether these cells could generate mature haemopoietic lineages. Equivalent numbers of blast-colony-derived cells, generated in the presence or absence of LY294002, were plated into HCAs in the absence of inhibitor. Erythroid and myeloid (granulocytic and macrophage) colonies did form from cells of blast-like colonies formed in the presence of LY294002 (Fig. 6A; EB 0LY/blast 5LY), confirming that differentiation had occurred within the blast colony as suggested by the gene expression analyses. However, there was a two- to threefold reduction in the number of colonies generated, indicating a reduction in the number of progenitors capable of differentiation in the blast colonies formed in the presence of LY294002. By contrast, inhibition of PI3Ks during the first 3.75 days of EB development, prior to re-plating in blast culture conditions (EB 5LY/blast 0LY), had no effect on the potential of blast-derived cells to generate haemopoietic colonies, confirming that PI3K inhibition during early EB development does not affect formation of the BL-CFC or its subsequent haemopoietic potential. Similar experiments could not be performed with PDK-1 deficient cells as too few progeny developed from the blast cultures (see Fig. 5C and D).

We also investigated whether PI3K signalling was required for the generation of terminally differentiated haemopoietic cells from haemopoietic progenitors present within day 6 EBs. Inhibition of PI3Ks only during the HCA cultures (see Fig. 6B) dramatically reduced the number of myeloid colonies formed and to a lesser extent the number of mixed colonies. Interestingly, in this condition the number of small primitive erythroid colonies did not alter; however, there appeared to be a decrease in the number of larger definitive erythroid colonies formed in the presence of LY294002. These definitive erythroid colonies were reduced in size (see inset, Fig. 6B) which may have contributed to the decreased number of colonies counted.

To further explore the effects of PI3K inhibition on the generation of terminally differentiated haemopoietic cells, we plated mouse primary bone marrow progenitors into HCA cultures in the presence or absence of LY294002. The majority of colonies that formed under our conditions were myeloid, with few erythroid and mixed colony types. Inhibition of PI3Ks reduced the total number of myeloid colonies two- to threefold, confirming the requirement for PI3K signalling on myeloid progenitor expansion. Interestingly, we noticed that LY294002 treatment led to a preferential reduction in GM colonies, whereas macrophage colony numbers were largely not affected, implying a specific role for PI3K signalling during formation of mature granulocytes. These data are consistent with the notion that PI3K-mediated signals play an important role in the differentiation and/or expansion of committed haemopoietic progenitors into mature myeloid lineage cells.

In this study we have used murine ES cells as an in vitro model of early embryonic differentiation and developmental haemopoiesis. Our investigations have revealed that PI3K-dependent signalling plays a major role in regulation of proliferation during EB-formation. Surprisingly, inhibition of PI3Ks during this earliest stage of development did not block the generation of progeny with haemopoietic potential, indicating that PI3K-signalling is not absolutely essential for their differentiation. Instead, when PI3K signalling was inhibited with LY294002, we observed an enhanced proportion of EB-derived cells capable of generating haemopoietic colonies, contrasting starkly with PDK1-deficient cells, which were unable to form mature erythroid or myeloid cells. These data indicate that PI3K-dependent signals, possibly mediated via PDK1, are required at a stage following haemopoietic progenitor formation within the EB for development of mature haemopoietic lineages. Investigation of the underlying mechanisms revealed an essential requirement for PI3K-mediated signals during expansion of BL-CFCs. Furthermore, we define a role for PI3K-dependent signalling during expansion of haemopoietic progenitors and generation of mature erythroid and myeloid lineages. These results, summarised in Fig. 7, support a role for PI3K-dependent signalling at multiple stages of developmental haemopoiesis.

Our data demonstrate a clear decrease in cellularity of EBs generated either in the presence of LY294002 or from PDK1-deficient ES cells. In our hands, this does not appear to be due to increased apoptosis (data not shown), which contrasts to the report that inhibition of PI3Ks leads to apoptosis in murine blastocysts and of trophoblast stem cells (Riley et al., 2006). However, it is worth noting that we used LY294002 at only 5 μM, compared with the 250 μM used by Riley et al. (Riley et al., 2006). It has been reported that LY294002 can also inhibit mTORC1 in vitro (Knight et al., 2006), although this occurs at higher doses of LY294002 than those required to inhibit class IA PI3Ks. Although we cannot formally discount the possibility that the more dramatic effects on EB cellularity we observe using 10 μM LY294002 are related to effects on mTORC1, we believe that the remarkably similar results obtained with ES cells treated with 5 μM LY294002 and PDK1-null ES cells are consistent with a role for class I PI3Ks during EB development. Our analyses of mesodermal and haemopoietic differentiation (cell surface marker and target gene expression) indicate that, despite the decrease in EB size, the overall proportion of mesodermal cells is not drastically altered and temporal patterns of gene expression are similar. Therefore, we believe our data are most consistent with PI3Ks being required for optimal proliferation of EB cells. The regulation of undifferentiated ES cell proliferation via PI3K-dependent signalling has been reported (Hallmann et al., 2003; Jirmanova et al., 2002; Paling et al., 2004; Sun et al., 1999; Takahashi et al., 2003) but very little is known about the involvement of PI3K signalling pathways during early stages of ES cell differentiation. Interestingly, S6 phosphorylation is increased in EB cells compared with undifferentiated ES cells (Fig. 1D), consistent with the increased PI3K activity seen following RA-induced differentiation of ES cells (Jirmanova et al., 2002) which could indicate increased dependence on this pathway as cells differentiate. The decrease in proliferation we propose to account for our observations is consistent with the consequences of PDK1 deficiency on embryonic mouse development, where PDK1 has been shown to be important for regulating growth (Lawlor et al., 2002). Similarly, deficiency of the p110β catalytic isoform of class IA PI3Ks leads to a very early embryonic lethality, with very poor growth of p110β-deficient blastocysts reported (Bi et al., 2002). Despite the fact that embryos as early as the one-cell stage express multiple class IA isoforms (Lu et al., 2004; Riley et al., 2005), this evidence suggests a non-redundant role for class IA PI3Ks. ES cells are derived from the inner cell masses of day 3.5 mouse blastocysts and development of EBs models the earliest stages of embryonic development. Thus, the effects we observe on EB development are consistent with a requirement for PI3K signalling during these early stages.

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 100× (10×/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 20× (10×/0.4 objective lens). Bars, 1 mm. In addition, colony morphologies are shown in the inserts; original magnification 100× (10×/0.4 objective lens). Bars, 200 μm. ***P<0.005 for GM colonies compared with 0 LY.

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 100× (10×/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 20× (10×/0.4 objective lens). Bars, 1 mm. In addition, colony morphologies are shown in the inserts; original magnification 100× (10×/0.4 objective lens). Bars, 200 μm. ***P<0.005 for GM colonies compared with 0 LY.

We were surprised to find that, despite the decrease in EB cellularity, the timing of early haemopoietic differentiation events - as evidenced by cell surface expression, gene expression analyses and formation of BL-CFCs - did not appear to be inhibited under conditions of PI3K inhibition or PDK1-deficiency, suggesting that PI3K signalling is dispensable for commitment towards the haemopoietic lineage during EB development. In fact, we observed an increase in the proportion of Flk-1+-Kit+ double-positive cells in day 6 EBs formed in the presence of LY294002. This Flk-1+-Kit+ double-positive population has been proposed to represent early haemopoietic progenitors (Lacaud et al., 2004; Willey et al., 2006), which correlates with our observed increase in haemopoietic-colony-forming potential of EB-derived cells generated in the presence of PI3K inhibition. Intriguingly, we appear to be detecting a distinct Flk-1high-Kit+ population in cells from day 6 EBs formed in the presence of LY294002 and, more prominently, in cells from EBs generated from PDK1-/- ES cells. It will be interesting to determine whether it is this distinct population that has haemopoietic potential. How can we explain the enhanced presence of cells with haemopoietic potential in EBs derived in the presence of PI3K inhibition? One possible explanation is that other cell types within the developing EB are differentially more sensitive to the growth inhibitory effects arising from PI3K inhibition, leading to a small increase in the total proportion of cells that have haemopoietic potential within the EB. This is certainly supported by our finding that PDK1-deficient ES cells also exhibit enhanced levels of a Flk-1+-Kit+ double-positive cell population. However, we cannot formally exclude the possibility that our results reflect either the inability of LY294002 to penetrate to the core of the developing EB, where the haemopoietic progenitors are likely to arise, or that an increase in the subset of cells capable of haemopoietic differentiation arises as a direct effect of PI3K inhibition during early development of these lineages.

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.

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.

The inability of PDK1-deficient EB-derived progeny to form terminally differentiated haemopoietic cells indicates a likely requirement for PDK1-mediated signals during developmental haemopoiesis. Our studies reveal a crucial requirement for PI3K signals during the expansion of the earliest haemopoietic progenitor, the BL-CFC, to form blast colonies. Quite remarkably, temporal expression of marker genes was not significantly altered, indicating that differentiation still occurs when PI3Ks are inhibited or PDK1 is absent. One caveat to these studies is that we have examined the behaviour of only a single PDK1-null ES-cell line, because an independently derived line was not available. However, the data generated with the PDK1-deficient cell line supports data generated with the PI3K inhibitor LY294002 and, together, are consistent with PI3K-mediated signals being required for proliferation of haemopoietic progenitors during blast colony expansion. This begins to shed light on the signals required for expansion and differentiation of the BL-CFC, about which little is known. VEGF, the ligand for Flk-1, plays a role in blast colony formation, although it is not known whether this involves PI3K signalling. However, PI3K signalling has been implicated in VEGF-mediated survival of murine bone marrow progenitors (Larrivee et al., 2003) and PI3Ks are activated by VEGF in adult human endothelial cells (Dayanir et al., 2001), so it will be interesting to examine this in the future. Consistent with a requirement for class IA PI3K-mediated signals in the expansion of haemopoietic progenitors, Kit+ haemopoietic progenitors derived from p85α-/- (Haneline et al., 2006; Huddleston et al., 2003) or p85α-/- p85β-/+ (Haneline et al., 2006; Huddleston et al., 2003) foetal liver show decreased proliferation in response to SCF.

We have also demonstrated a role for PI3K-dependent signalling in the generation of committed haemopoietic precursors. Several studies have suggested a role for PI3K signalling during foetal liver erythropoiesis (Huddleston et al., 2003; Zhao et al., 2006) and recent work has demonstrated a requirement for the PI3K/AKT signalling pathway in the maturation of foetal liver erythroid progenitors (Ghaffari et al., 2006). Our data are consistent with a requirement for PI3K signalling for expansion of haemopoietic progenitors into the mature erythroid lineage. In addition, we demonstrate that inhibition of PI3Ks only during expansion of committed progenitors dramatically reduced the number of myeloid colonies formed from haemopoietic progenitors derived from both EBs and mouse bone marrow. Although there have been reports of many cytokines activating PI3Ks (Gold et al., 1994) and of PI3Ks being involved in regulation of proliferation of mature myeloid lineages (Ali et al., 2004; Fox et al., 2005; Mungalavadla et al., 2005), there is little data available on the role of PI3Ks in development of the myeloid lineage. During the preparation of this manuscript, a requirement for class IA PI3Ks in the function of foetal liver-derived haemopoietic stem cells was reported (Haneline et al., 2006), which demonstrated significantly reduced myeloid repopulating activity of p85α-/- / p85β-/+ adult HSCs. These studies, together with our data, are consistent with PI3Ks contributing to differentiation of the myeloid lineage.

We have shown that PI3K-dependent signalling events, mediated at least in part via PDK1, are required for proliferation during early differentiation within EBs and, therefore, may be required for overall proliferation events during early development. Importantly, PI3K signalling pathways are required for efficient expansion of BL-CFC progeny, as well as for optimal differentiation of erythroid and myeloid progenitors, indicating important roles for these signals at multiple stages of haemopoietic development.

Embryonic stem cell growth and differentiation

The E14tg2a wild-type and PDK1-deficient murine ES cell lines (Williams et al., 2000; a kind gift from Dario Alessi, University of Dundee, UK) were routinely cultured in knock-out Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 103 units/ml murine LIF (ESGRO; Chemicon) as described previously (Paling and Welham, 2005; Paling et al., 2004). Formation of embryoid bodies (EBs) was adapted from Kennedy et al. (Kennedy et al., 1997). ES cells were trypsinized into a single-cell suspension and plated at 1×104 cells/ml in 40% (v/v) ES-Cult (StemCell Technologies, Vancouver, Canada) containing 15% (v/v) FBS (Invitrogen), 0.2 mg/ml transferrin (Sigma), 4.5×10-4 M monothioglycerol (MTG, Sigma), 50 μg/ml L-ascorbic acid, and 100 μg/ml insulin in Iscove's modified Dulbecco's medium (IMDM, Invitrogen) supplemented with 10 ng/ml basic fibroblast growth factor (bFGF) (PeproTech, UK) and 2 ng/ml activin A (recombinant human, R&D Systems) and plated into bacterial Petri dishes (Sterilin) in the presence or absence of 5 or 10 μM LY294002 (Calbiochem).

Colony assays

EBs were harvested, washed in PBS (Invitrogen) and trypsinized for 3 minutes at 37°C. Blast colonies were formed by plating day 3 to day 4 EB-derived cells at 3-5×104 cells/ml in 40% (v/v) ES-Cult (StemCell Technologies) containing 10% (v/v) FBS (Invitrogen), 0.2 mg/ml transferrin, 4.5×10-4 M MTG, 25 μg/ml L-ascorbic acid in IMDM supplemented with 5 ng/ml vascular endothelial growth factor (VEGF; PeproTech), 5 ng/ml IL-6 (Peprotech) and 25% D4T endothelial cell conditioned medium (Kennedy et al., 1997). Colonies were counted following 4 days in culture. For the growth of haemopoietic precursors, day 6 EB-derived cells were plated at 5-10×104 cells/ml in Metho-cult (StemCell Technologies) containing 1% (w/v) methylcellulose 15% (v/v) FBS, 1% (w/v) bovine serum albumin (BSA), 10 μg/ml insulin 0.2 mg/ml transferrin, 1×10-4 M 2-mercaptoethanol and 2 mM glutamine, supplemented with 25ng/ml GM-CSF, 25 ng/ml G-CSF, 10 ng/ml IL-3, 10 ng/ml SCF (all from Peprotech) and 2 U/ml erythropoietin (EPO: R&D Systems) in IMDM and plated into 35-mm Petri dishes. Alternatively, bone marrow (BM) was harvested from the femurs of 12-week-old CD1 mice, red blood cells cleared by hypotonic lysis, and plated at 2.5×104 cells/ml in above culture conditions. Primitive erythroid colonies were counted following 4-5 days in culture and appeared as very small red clusters of 10-20 erythroblasts. All other colonies were counted following 8-10 days in culture. Colony images were visualised using an Olympus (Tokyo, Japan) XI51 inverted microscope and objective lenses (apertures and magnifications are specified in each figure legend) and captured using an Olympus Camedia C4040 digital camera. Data were analysed for statistical significance using two-tailed paired Student's t-tests.

Cell lysates and immunoblotting

EBs were washed three times in PBS and lysed directly in ice-cold solubilisation buffer, as described previously (Welham et al., 1994). Insoluble material was removed by centrifugation at full speed for 2 minutes in an Eppendorf microcentrifuge at 4°C. Protein concentrations were determined using the Bio-Rad (Hercules, CA) protein assay kit according to the manufacturer's instructions and 10 μg of each cell lysate separated by SDS-PAGE an transferred onto nitrocellulose (Welham et al., 1994). Immunobloting was performed using primary antibodies at 1:1000 dilution: rabbit polyclonal antibodies recognising double phosphorylated ribosomal protein S6 at Ser235-Ser236 (anti-P-S6, Cell Signal Technologies, 2211) or SHP-2 (Santa Cruz Biotechnology) or goat polyclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz, sc-20357). Anti-rabbit and anti-goat secondary antibodies conjugated to horseradish peroxidase (Dako, UK), were used at 1:10 000 dilution and blots were developed using ECL (Amersham Biosciences, UK). Blots were stripped and re-probed as described previously (Welham et al., 1994).

Flow cytometry

EBs were trypsinised and the cells resuspended in ice-cold wash buffer [PBS containing 2% (v/v) FBS and 0.1% (w/v) sodium azide]. Non-specific binding to Fc receptors was blocked by incubation with mouse anti-CD16/CD32 anibody (FcγIII/II receptors; 1:2500; BD PharMingen) for 30 minutes on ice. Cells were then stained on ice for a further 30 minutes with phycoerythrin (PE)-conjugated anti-Kit monoclonal antibody (clone ACK45;1:200; BD PharMingen) and/or biotin-conjugated anti-Flk-1 monoclonal antibody (clone Avas12a1; 1:100; eBioscience) in ice-cold wash buffer. Flk-1-labeled cells were then washed twice in wash buffer and incubated with (PE-Cy5)-conjugated streptavidin (1:100; DAKO) for a further 30 minutes. Flow cytometry was performed using a FACSCanto cytometer (Becton Dickenson) and the data were analysed with FACSDiva software. Dead cells were excluded from analyses based on forward and side scatter parameters.

Gene expression analyses

EBs were collected, washed in PBS and RNA was purified using TRIzol reagent (Invitrogen), following the manufacturer's instructions. All RNA samples were treated with DNase I (Promega) before cDNA synthesis to eliminate any contaminating genomic DNA. RNA (1 μg) was reverse transcribed into cDNA with Oligo(dT)15 (Promega) using SuperScript II (Invitrogen). Gene-specific PCR was carried out using the following primers (Faloon et al., 2000; Keller et al., 1993; Ogawa et al., 1999): brachyury sense, 5′-CATGTACTCTTCTTGCTGG-3′; brachyury antisense, 5′-GGTCTCGGGAAAGCAGTGGC-3′; Kit sense, 5′-TGTCTCTCCAGTTTCCCTGC-3′; Kit antisense, 5′-TTCAGGGACTCATGGGCTCA-3′; flk1 sense, 5′-CACCTGGCACTCTCCACCTTC-3′; flk1 antisense, 5′-GATTTCATCCCACTACCGAAAG-3′; scl sense, 5′-ATTGCACACACGGGATTCTG-3′; scl antisense, 5′-GAATTCAGGGTCTTCCTTAG-3′; gata1 sense, 5′-ACTCGTCATACCACTAAG-3′; gata1 antisense, 5′-AGTGTCTGTAGGCCTCAG-3′; β-major sense, 5′-CGATGAAGTTGGTGGTGAGG-3′; β-major antisense, 5′-CACAATCACGATCATATTGCC-3′; β-H1 sense, 5′-AGTCCCCATGGAGTCAAAGA-3′; β-H1 antisense, 5′-CTCAAGGAGACCTTTGCTCA-3′; gapdh sense, 5′-TGGAGCCAAACGGGTCATC-3′; gapdh antisense, 5′-GCTCTGGGATGACCTTGCC-3′; β-actin sense, 5′-TAGGCACCAGGGTGTGATGG-3′; β-actin antisense, 5′-CATGGCTGGGGTGTTGAAGG-3′.

Quantitative PCR was performed using a two-step RT-PCR method and LightCycler FastStart DNA Master SYBR Green I (Roche Applied Science) according to the manufacturer's instructions using the above primers. Briefly, the reaction was carried out in a total volume of 20 μl, comprising 0.5 μM of each primer, 2.5 mM MgCl2, 2 μl SYBR Green, and 2 μl of appropriately diluted cDNA (prepared as described above). Subsequent amplification and online monitoring was performed using the LightCycler™ system (Roche Applied Science). After 40 cycles of amplification, melting-curve analysis was performed to check that only the desired PCR product had been amplified. All target gene levels were normalised to that of β-actin for each sample. The PCR efficiency of both the target and reference genes was calculated from the derived slopes of standard curves by the LightCycler software (Roche Molecular Biochemicals LightCycler Software, Version 3.5.3). These PCR efficiency values were used to calculate the relative quantification values for calibrator-normalised target gene expression by the LightCycler Relative quantification software (Version 1.0). Data were analysed for statistical significance using two-tailed paired Student's t-tests.

We thank Helen Wheadon and Nick Paling for initial assistance with ES differentiation, Dario Alessi for PDK1-/- ES cells, and George Lacaud and Gordon Keller for D4T cell lines. This work was funded by the Wellcome Trust (grant reference 070316/Z/03/Z).

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