1752 Research Article

IntroductionPhosphoinositide 3-kinases (PI3Ks) are a family of lipidkinases, whose products, phosphatidylinositol (3,4)-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3], act as intracellular secondmessengers (Cantley, 2002; Vanhaesebroeck et al., 2001;Vanhaesebroeck and Waterfield, 1999). Three classes of PI3Khave been described based on their structure and in vitrosubstrate specificity (Vanhaesebroeck and Waterfield, 1999).Members of the class IA family of PI3Ks are composed of aregulatory subunit (p85 or p55) and a p110 catalytic subunit(�, � or �), and are activated by numerous growth factors andcytokines (Cantley, 2002; Vanhaesebroeck et al., 2001;Vanhaesebroeck and Waterfield, 1999). 3-phosphoinositide-dependent protein kinase 1 (PDK1, also known as PDPK1) isa direct downstream effector of PI3Ks (Vanhaesebroeck andAlessi, 2000) and plays a central role in activating otherdownstream AGC-family kinases, ultimately leading to theregulation of a wide array of physiological processes, includingproliferation, survival and differentiation (Mora et al., 2004).Gene-targeting studies in mice, in which one or more of theclass IA PI3K sub-units has been functionally disrupted, haverevealed a requirement for this family of enzymes in regulatingdevelopmental events. For example, disruption of both p85�and p85� gene products results in lethality at embryonic day12.5 (E12.5) (Brachmann et al., 2005), whereas embryosdeficient in p110� die between E9.5 and E10.5 due to aproliferative defect (Bi et al., 1999). Importantly, deletion ofp110� leads to a very early developmental defect, resulting in

embryonic lethality at the pre-implantation blastocyst stage (Biet al., 2002). Interestingly, class IA catalytic and regulatorysubunits are expressed from the single-cell stage ofdevelopment (Lu et al., 2004; Riley et al., 2005) and PI3Ksignalling appears to be required for correct development ofpre-implantation mouse embryos (Lu et al., 2004; Riley et al.,2005; Riley et al., 2006). The phenotypes of several PI3Kknockout mice have highlighted a requirement for PI3K-mediated signalling in development of the haemopoieticsystem. Disruption of p85� alone has demonstrated a role forPI3Ks in erythropoiesis (Huddleston et al., 2003), whereasp85�–/–/p85�–/+ foetal liver-derived haemopoietic stem cellshave recently been reported to have reduced repopulatingability (Haneline et al., 2006). Both p85� and the p110�subunits have been shown to be involved in B and T celldevelopment (Clayton et al., 2002; Fruman et al., 1999;Okkenhaug et al., 2002; Suzuki et al., 1999). These reportssuggest that PI3Ks play an important role during earlyembryonic development and that PI3Ks may be specificallyrequired during developmental haemopoiesis.

The inaccessibility of early embryonic material and theembryonic lethality of several PI3K gene knockouts have madeit 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 thecontribution of specific signals to early developmental stagescan be examined (Doetschman et al., 1985; Keller et al., 1993;Keller, 1995). Extensive genetic analyses have demonstratedthat in vitro differentiation of ES cells into embryoid bodies

Phosphoinositide 3-kinase (PI3K)-dependent signallingregulates a wide variety of cellular functions includingproliferation and differentiation. Disruption of class IA PI3Kisoforms has implicated PI3K-mediated signalling indevelopment of the early embryo and lymphohaemopoieticsystem. We have used embryonic stem (ES) cells as an invitro model to study the involvement of PI3K-dependentsignalling during early development and haemopoiesis. Bothpharmacological inhibition and genetic manipulation ofPI3K-dependent signalling demonstrate that PI3K-mediatedsignals, most likely via 3-phosphoinositide-dependentprotein kinase 1 (PDK1), are required for proliferation ofcells within developing embryoid bodies (EBs). Surprisingly,the haemopoietic potential of EB-derived cells was notblocked upon PI3K inhibition but rather enhanced,

correlating with modest increases in expression ofhaemopoietic marker genes. By contrast, PDK1-deficientEB-derived progeny failed to generate terminallydifferentiated haemopoietic lineages. This deficiencyappeared to be due to a requirement for PI3K signallingduring the proliferative phase of blast-colony-forming cell(BL-CFC) expansion, rather than as a result of effects ondifferentiation per se. We also demonstrate that PI3K-dependent signalling is required for optimal generation oferythroid and myeloid progenitors and their differentiationinto mature haemopoietic colony types. These datademonstrate that PI3K-dependent signals play importantroles at different stages of haemopoietic development.

Key words: PI3K; Embryoid bodies, Differentiation

Summary

Phosphoinositide 3-kinase signalling regulates earlydevelopment and developmental haemopoiesisHeather K. Bone and Melanie J. Welham*Department of Pharmacy and Pharmacology and the Centre for Regenerative Medicine, University of Bath, Claverton Down, Bath, BA2 7AY, UK*Author for correspondence (e-mail: M.J.Welham@bath.ac.uk)

Accepted 29 April 2007Journal of Cell Science 120, 1752-1762 Published by The Company of Biologists 2007doi:10.1242/jcs.003772

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1753PI3Ks regulate developmental haemopoiesis

(EBs) recapitulates the events leading to the onset of primitivehaemopoiesis in the yolk sac of the normal mouse embryo(Keller, 2005; Keller et al., 1993; Palis et al., 1999). Using EScell differentiation, a progenitor representing the earliest stageof 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 bothhaemopoietic and endothelial potential and can becharacterised by co-expression of Flk-1 (also known as Kdr)and the mesodermal gene brachyury (Choi et al., 1998; Faloonet al., 2000; Fehling et al., 2003; Kennedy et al., 1997). TheBL-CFC represents the in vitro equivalent of the yolk sachaemangioblast, recently identified in the early mouse embryo(Huber et al., 2004).

To further examine the role of PI3K-dependent signallingpathways during early development and, more specifically,developmental haemopoiesis, we have used ES cells as an invitro model and employed both pharmacological and geneticapproaches to manipulate PI3K activity at different stages ofdifferentiation. LY294002 is a reversible inhibitor (in vitro IC501.4 �M) of all classes of PI3K (class IA, IB, II and III), althoughthe activity of PI3K-C2� is refractory to concentrations ofLY294002 that inhibit the activity of the other PI3K familymembers (Knight et al., 2006; Vlahos et al., 1994). LY294002is stable in solution and can be added or removed from assaysat various points, making it a useful tool for deciphering thebiological requirement of PI3K signalling at a cellular level.To complement the inhibitor studies and to investigate the roleof class IA PI3Ks specifically, we employed ES cells in whichPDK1, a crucial downstream effector of class IA PI3Ks, hasbeen disrupted. Using these complementary approaches, wedemonstrate that the major role of PI3Ks during the earlieststages of ES cell differentiation is to regulate proliferation. Atlater stages of differentiation we demonstrate that PI3K-dependent signalling is required for expansion of BL-CFC andhaemopoietic precursors. Thus, PI3Ks play multiple roles atdefined times during early development and differentiation ofthe primitive haemopoietic system.

ResultsPI3K-dependent signalling is required for optimal EBdevelopmentInitially, we examined whether inhibition of PI3K signallingaffects the ability of ES cells to develop into EBs. ES cellswere differentiated into EBs in the absence or presence of 5or 10 �M of the PI3K inhibitor LY294002 (Vlahos et al.,1994). As seen in Fig. 1A, the size of EBs decreased uponinhibition of PI3K signalling, correlating with a two- tothreefold reduction in cellularity in the presence of 5 �MLY294002 (see Fig. 1B), whereas 10 �M LY294002 almostcompletely inhibited EB development. Importantly, despitethe size reduction, inhibition of PI3Ks with 5 �M LY294002did not affect the plating efficiency of the ES cells, the samenumber of EBs being generated irrespective of the presenceof the PI3K inhibitor (Fig. 1C). In addition, the decrease incellularity 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 wemeasured the status of S6 phosphorylation at serine residuesSer235 and Ser236 by immunoblotting (Fig. 1D). S6phosphorylation was significantly reduced in EBs treated

with 5 �M LY294002 and almost completely inhibited with10 �M LY294002.

LY294002 inhibits all classes of PI3K, with the exceptionof PI3K-C2� (Knight et al., 2006; Vlahos et al., 1994) andwe wanted to investigate more specifically the role of classIA PI3K. However, the complexity of this family (seeIntroduction) precluded a genetic knock-out approach of all

Fig. 1. Inhibition of PI3Ks impairs EB formation. EBs weregenerated in the presence of 0, 5 and 10 �M LY294002 (LY) asdescribed in Materials and Methods. (A) Examples of day 5 EBs,original magnification 100� (10�/0.4 objective lens was used).Bars, 200 �m. (B) On day 3 to day 6 samples of EBs weretrypsinized and individual cells counted. Data shown are mean (±s.e.m.) from five independent experiments. (C) The total number ofEBs formed were counted on day 6. Data shown are mean (± s.e.m.)from four independent experiments. (D) Protein extracts wereprepared from ES cells (ES) and EBs on the days indicated andseparated by SDS-PAGE. Immunoblots were probed with anantibody detecting phosphorylated serine residues Ser235 andSer236 of the ribosomal S6 protein (�-P-S6). The same immunoblotwas stripped and reprobed for SHP-2 to assess equal protein loading.*P<0.05, **P�0.01, ***P<0.005.

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isoforms within the same ES cell. Therefore, we reasoned thatES cells in which PDK1, a crucial downstream effector ofclass I PI3K, had been disrupted would provide a robustgenetic tool to determine whether the effects we observe withLY294002 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 atwo- to threefold decrease in cellularity compared with wild-type parental ES cells (Fig. 2A,B), similar to the effectsobserved with 5 �M LY294002 (Fig. 1). The platingefficiency of the PDK1–/– ES cells was not significantlydifferent from wild-type ES cells (Fig. 2C). S6phosphorylation could not be detected by immunoblottingextracts from PDK1–/– EBs (Fig. 2D), indicating that PDK1is required in the pathway leading to phosphorylation of S6,as previously reported (Williams et al., 2000). These dataindicate that PI3K signalling pathways, mediated at least inpart via PDK1, are required for optimal formation anddevelopment of EBs and are consistent with class IA PI3Ksplaying a role in early developmental processes.

Inhibition of PI3K-dependent signalling does not blockearly developmental haemopoiesis but rather enhancesthe haemopoietic potential of EB-derived cellsOur results demonstrate that PI3K-dependent signalling eventsare required for optimal development of EBs. We wereinterested in determining whether haemopoietic differentiationhad been affected when PI3K signalling was perturbed duringEB formation. To detect the emergence of haemopoieticprecursors within the developing EB, EBs were formed in thepresence or absence of LY294002 and we examined the abilityof 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 maturationof a range of haemopoietic cell types. Somewhat surprisingly,inhibition of PI3Ks during EB development led to an increasein the proportion of EB progeny with haemopoietic potential,evidenced by increases in the number of erythroid, myeloidand mixed colonies present in the HCAs compared withuntreated EBs (Fig. 3A). Few secondary EBs developed,indicating efficient differentiation of cells within the EBs. Bycontrast, when we performed similar experiments with day 6PDK1-deficient EB-derived cells, only very small erythroidcolonies formed (Fig. 3B), whereas by day 12 no colonies werevisible. Contrary to inhibition of PI3Ks with LY294002 onlyduring EB formation (Fig. 3A), which measures the effects ofPI3K inhibition on progenitor formation within the EB, in thisexperiment using PDK1–/– ES cells, the class I PI3K pathwaywould be inhibited throughout the entire experiment, includingduring formation of colonies in the HCAs. These resultssuggest that PDK1-dependent signals are required duringdevelopmental haemopoiesis, following haemopoieticprogenitor formation within the EB. We were interested infurther defining the stage at which PI3K signalling wasrequired.

The emergence of different ES-cell-derived haemopoieticpopulations within the developing EB can be tracked byexpression of specific cell surface markers (Kabrun et al.,1997; Mikkola et al., 2003; Nishikawa et al., 1998). Flk-1 isinitially expressed on BL-CFCs and represents the onset of

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embryonic haemopoiesis (Faloon et al., 2000; Fehling et al.,2003; Kabrun et al., 1997), whereas the proto-oncogeneproduct Kit marks cells committed to the haemopoietic lineageand 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 haemopoieticpotential that mark the establishment of haemopoiesis (Lacaudet al., 2004; Willey et al., 2006). The pattern of Flk-1 and Kitsurface expression on cells from developing EBs formed in thepresence or absence of 5 �M LY294002 or formed fromPDK1–/– ES cells was analysed by flow cytometry. In untreated

Fig. 2. EBs formed from PDK1–/– ES cells are reduced in size andcellularity. EBs were generated from wild-type parental (WT) andPDK1–/– ES cells. (A) Examples of day 5 EBs, original magnification100� (10�/0.4 objective lens was used). Bars, 200 �m. (B) On day 3to day 6, samples of EBs were trypsinised and the cells counted. Datashown are the mean (± s.e.m.) from two to three independentexperiments. (C) The mean number of EBs formed, counted on day 6(± s.e.m.) from three independent experiments. (D) Protein extractsfrom EBs formed from wild-type (WT) or PDK1–/– (ko) ES cells wereprepared on the days indicated, separated by SDS-PAGE andimmunoblotted with antibody against phosphorylated S6 (�-P-S6).The blot was then stripped and reprobed for GAPDH. *P<0.05,***P<0.005.

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wild-type EBs, little Flk-1 or Kit was expressed on day 3(results not shown). As shown in Fig. 3C, expression of Flk-1dramatically increased over the next 24 hours followed by theexpression of Kit, which was maximal on day 5 and day 6.Importantly, a Flk-1+-Kit+ double-positive population wasobserved 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 PI3Kswith 5 �M LY294002 led to no change in overall Kitexpression, although a small enhancement in overall Flk-1expression 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 withuntreated EBs, correlating with the enhanced formation ofhaemopoietic colonies observed (Fig. 3B). Despite not beingable to generate mature haemopoietic colonies, cells fromPDK1–/– EBs showed an increase in overall Flk-1 expression,culminating in an enhanced Flk-1+-Kit+ double-positive cellpopulation (Fig. 3C). These findings suggest that inhibition ofPI3K signalling does not block formation of earlyhaemopoietic progenitors and may result in an enhancedproportion of EB cells with haemopoietic potential.

We also examined expression of marker genes to trackprogression from the mesoderm (brachyury) to the BL-CFC

(Flk-1) and on to committed haemopoietic progenitors (Scl andGata-1) (Chung et al., 2002; D’Souza et al., 2005; Fehling etal., 2003; Fujimoto et al., 2001; Robertson et al., 2000), as wellas the globin genes �-H1 (primitive erythroid) and �-major(definitive erythroid). Using semi-quantitative RT-PCR (Fig.4A and B) inhibition of PI3K-dependent signalling byLY294002 treatment or disruption of PDK1 did not appear tosignificantly affect either the timing or apparent magnitude ofexpression of this set of marker genes. However, such semi-quantitative approaches cannot adequately distinguishrelatively modest changes in gene expression and in light ofthe results of our HCAs, we decided to measure expression ofthese genes by quantitative PCR. Expression of brachyury wasreduced in day 3 EBs formed in the presence of LY294002(Fig. 4C) or derived from PDK1–/– ES cells (Fig. 4D). Despitethe increased surface expression of Flk-1 in EBs formed in thepresence of LY294002 or from PDK1–/– ES cells, Flk-1 geneexpression was reduced in day 4 EBs with a slightenhancement by day 6 in EBs formed in the presence ofLY294002 (Fig. 4C and D). In light of the enhancedhaemopoietic potential of EB cells derived in the presence ofLY294002 observed in our HCAs, we were particularlyinterested in examining the expression of Scl, Gata-1, �-H1and �-major. By day 6, all were upregulated two- to threefold

Fig. 3. Inhibition of PI3K-dependentsignalling does not block haemopoiesisin early development but, rather,enhances it. (A) The number ofhaemopoietic colonies formed by day 6EB-derived cells differentiated in theabsence (EB 0LY/HCA 0LY) orpresence of 5 �M LY294002 (EB5LY/HCA 0LY). Colonies were definedas follows: primitive erythroid (Ery/P),definitive erythroid (Ery/D), myeloid(including macrophage andgranulocyte/macrophage colonies),mixed lineage (including erythroid-macrophage colonies and multi-lineagecolonies), and secondary EBs (EB).Data are the mean of quadruple counts(± s.e.m.) from two independentexperiments, *P<0.05; **P�0.01.(B) Images of day 6 erythroid colonies(left panels) and day 12 haemopoieticcolonies (right panels) formed by day 6WT or PDK1–/– EB cells [originalmagnification 100� (left panels) or20� (right panels)]. Bars, 400 �m.Arrow indicates erythroid colony.(C) FACS analysis of Kit (c-kit) andFlk-1 expression of cells derived fromEBs formed in the absence (0 �M LY)or presence (5 �M LY) of 5 �MLY294002 or formed from PDK1–/– EScells. The days of differentiation areindicated. Numbers in each quadrantrepresent the percent of the total livecells in each sample. Similar trendswere observed in three independentexperiments.Jo

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in EBs formed in the presence of LY294002, correlating withthe enhanced haemopoietic potential of these day 6 EB-derivedcells. Taken together, these data indicate that inhibition ofPI3K-dependent signalling does not block early stages ofhaemopoietic differentiation within developing EBs.

PI3K-dependent signalling is required for expansion ofBL-CFC progenyHaving observed that inhibition of PI3K-dependent signallingwithin developing EBs does not cause gross changes in theexpression of differentiation markers but absence of PDK1appears to preclude development of terminally differentiated

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haemopoietic cells, we decided to examine whether PI3K-mediated signalling was involved in the formation orexpansion of BL-CFCs, the earliest haemopoietic progenitors.BL-CFCs are transient progenitors that arise in EBs betweenday 2.5 and day 4 of differentiation, and are able to generateblast 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-1and brachyury (Faloon et al., 2000; Fehling et al., 2003;Kabrun et al., 1997). We first determined whether inhibitingPI3Ks affected differentiation towards BL-CFCs within theEBs by assessing the ability of EB-derived cells to form blast

Fig. 4. Inhibition of PI3K-dependent signalling enhances expression of haemopoietic marker genes. (A) RT-PCR analysis of wild-type EBsformed in the absence (0 LY) or presence of 5 �M 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) individualexperiments. In A the samples for Scl, Gata-1, �-H1 and �-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 �M LY d3 samples. (C,D) Quantitative RT-PCRanalysis showing relative levels of the indicated RNA normalised to �-actin. (C) The average and s.d. of three to four replicates from the sameexperimental RNA as in A are shown and are representative of three independent experiments. White bars, RNA from EBs generated in theabsence of LY294002; black bars, RNA samples from EBs generated in the presence of 5 �M LY294002. (D) The mean and ±s.e.m. ofquadruple 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.

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colonies. EBs generated in the presence or absence ofLY294002 were harvested following 3, 3.75 and 4 days ofdifferentiation, and identical cell numbers were plated underblast-colony-forming conditions with no further inhibitoradded. As indicated in Fig. 5A and B, cells from day 3 EBswere 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 withexpression of Flk-1 (Fig. 3C) and brachyury (Fig. 4A).Culturing EBs with 5 �M LY294002 had little or no affect onthe number of blast colonies formed (Fig. 5B) or theircellularity. These data demonstrate that inhibition of PI3Ksdoes not block differentiation towards BL-CFCs within EBs.However, when we performed similar experiments withPDK1–/– ES cells no blast colonies formed (Fig. 5C). This wasnot simply due to a temporal change in developmentalpotential, 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 theEB. However, we feel this is unlikely because these cellsexpress Flk-1 and brachyury in a pattern similar to wild-typeparental ES cells (Fig. 3C and Fig. 4B). (2) PI3K signals,mediated at least in part via PDK1, are required for theexpansion and/or differentiation of the BL-CFC to form a blastcolony. We tested this possibility by assessing whetherinhibiting PI3Ks only during blast culture affected blast colonyformation. Cells from EBs, formed in the absence of inhibitorfor 3.75 days to allow for formation of the BL-CFC, wereplated under blast-colony-forming conditions in the presenceor absence of 5 �M LY294002. Inhibition of PI3K signallingwith 5 �M LY294002 significantly inhibited the ability of BL-CFCs to form archetypal blast colonies – only small clumps ofcells were observed (Fig. 5C) – corresponding to a tenfolddecrease in cellularity (Fig. 5D). These data indicate that PI3K-dependent signalling is required for BL-CFCs to undergoexpansion to form blast colonies.

Blast colony formation has been described as a sequentialprocess involving both proliferation and differentiation events(D’Souza et al., 2005). Within 1-2 days in culture the BL-CFCfirst proliferates to form a tight core of cells. Haemopoietic

Fig. 5. PI3K-dependent signalling is required for formation of blastcolonies. (A) EBs were formed in the presence of 0 or 5 �MLY294002 (LY). EB cells were harvested on day 3, 3.75 or 4 asindicated and identical cell numbers (5�104/ml) plated under blastculture conditions. Examples of blasts cultured for 4 days; originalmagnification 100� (10�/0.4 objective lens was used). Bars, 200�m. (B) Following 4 days in culture, the blast colonies werecounted. Data represent the average of triplicate counts (± s.d.) andare representative of four independent experiments. (C) EBs wereformed from either untreated wild-type or PDK1–/– ES cells and cellswere harvested following 3.75 days in culture. Identical cell numbers(3�104/ml) were then plated under blast culture conditions. Cellsfrom EBs generated from the wild-type ES cells were plated either inthe absence (0 LY) or the presence of 5 �M LY294002. Examples ofblasts cultured for 4 days; original magnification 100� (10�/0.4objective lens was used). Bars, 200 �m. (D) Following 4 days inculture the blast colonies were dissociated and the total number ofcells counted. Data represent mean (± s.e.m.) from three individualexperiments. **P�0.01 (E and F) RT PCR analyses of 2° blasts.Wild-type or PDK1–/– day 3.75 EB-derived cells were plated underblast culture conditions for 2-4 days. Cells from EBs generated fromuntreated wild-type ES cells were plated in the absence (0 LY) orpresence of 5 �M LY294002 (5 �M LY). RNA was isolated from theinitial day 3.75 EB cells (EB) and from the resulting blasts (days ofdifferentiation are indicated) and used for RT-PCR analysis of themarker genes depicted. Note that the EB sample for Scl in E appearsin the second lane.

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cells then develop from the core and after 3-4 days these cellscover the core. Haemopoietic, endothelial and vascular smoothmuscle lineages comprise the mature blast colony. Our resultscould either be due to effects on proliferation – directlyaffecting growth of the blast colony – or due to effects ondifferentiation. To distinguish between these two possibilities,we examined the temporal expression of marker genes to trackblast colony differentiation. As shown in Fig. 5E, patterns ofgene expression were very similar in blast colonies generatedwhen PI3K was inhibited or not, with flow cytometrydemonstrating similar cell-surface-expression profiles forFlk-1, Kit and CD41 (data not shown). Intriguingly, despite nooutward appearance of PDK1-deficient EB-derived cells beingable to form blast colonies, cells recovered from these culturesshowed a characteristic downregulation of brachyury as wellas expression of genes encoding Scl, Gata-1, �-H1 and �-major (Fig. 5F). These data indicate that there are no dramaticalterations in the blast colony differentiation profiles in theabsence of PI3K-dependent signalling; therefore, in light of thedecline in size of blast colonies, the most likely explanation ofour results is that PI3K-dependent signals are required forproliferation of progenitor cells within the developing blastcolony.

PI3K-dependent signalling is required for optimaldevelopment of haemopoietic precursors and maturehaemopoietic lineagesOur results indicate that PI3K-mediated signals are requiredfor expansion of the BL-CFC to form a blast colony. However,analyses of marker gene expression indicated thathaemopoietic differentiation can still occur within the cellswhich do develop, so we decided to investigate whether thesecells could generate mature haemopoietic lineages. Equivalentnumbers of blast-colony-derived cells, generated in thepresence or absence of LY294002, were plated into HCAs inthe absence of inhibitor. Erythroid and myeloid (granulocyticand macrophage) colonies did form from cells of blast-likecolonies formed in the presence of LY294002 (Fig. 6A; EB0LY/blast 5LY), confirming that differentiation had occurredwithin the blast colony as suggested by the gene expressionanalyses. However, there was a two- to threefold reduction inthe number of colonies generated, indicating a reduction in thenumber of progenitors capable of differentiation in the blastcolonies formed in the presence of LY294002. By contrast,inhibition of PI3Ks during the first 3.75 days of EBdevelopment, prior to re-plating in blast culture conditions (EB5LY/blast 0LY), had no effect on the potential of blast-derivedcells to generate haemopoietic colonies, confirming that PI3Kinhibition during early EB development does not affectformation of the BL-CFC or its subsequent haemopoieticpotential. Similar experiments could not be performed withPDK-1 deficient cells as too few progeny developed from theblast cultures (see Fig. 5C and D).

We also investigated whether PI3K signalling was requiredfor the generation of terminally differentiated haemopoieticcells 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 formedand to a lesser extent the number of mixed colonies.Interestingly, in this condition the number of small primitiveerythroid colonies did not alter; however, there appeared to be

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a decrease in the number of larger definitive erythroid coloniesformed in the presence of LY294002. These definitiveerythroid colonies were reduced in size (see inset, Fig. 6B)which may have contributed to the decreased number ofcolonies counted.

To further explore the effects of PI3K inhibition on thegeneration of terminally differentiated haemopoietic cells, weplated mouse primary bone marrow progenitors into HCAcultures in the presence or absence of LY294002. The majorityof colonies that formed under our conditions were myeloid,with few erythroid and mixed colony types. Inhibition ofPI3Ks reduced the total number of myeloid colonies two- tothreefold, confirming the requirement for PI3K signalling onmyeloid progenitor expansion. Interestingly, we noticed thatLY294002 treatment led to a preferential reduction in GMcolonies, whereas macrophage colony numbers were largelynot affected, implying a specific role for PI3K signallingduring formation of mature granulocytes. These data areconsistent with the notion that PI3K-mediated signals play animportant role in the differentiation and/or expansion ofcommitted haemopoietic progenitors into mature myeloidlineage cells.

DiscussionIn this study we have used murine ES cells as an in vitro modelof early embryonic differentiation and developmentalhaemopoiesis. Our investigations have revealed that PI3K-dependent signalling plays a major role in regulation ofproliferation during EB-formation. Surprisingly, inhibition ofPI3Ks during this earliest stage of development did not blockthe generation of progeny with haemopoietic potential,indicating that PI3K-signalling is not absolutely essential fortheir differentiation. Instead, when PI3K signalling wasinhibited with LY294002, we observed an enhanced proportionof EB-derived cells capable of generating haemopoieticcolonies, contrasting starkly with PDK1-deficient cells, whichwere unable to form mature erythroid or myeloid cells. Thesedata indicate that PI3K-dependent signals, possibly mediatedvia PDK1, are required at a stage following haemopoieticprogenitor formation within the EB for development of maturehaemopoietic lineages. Investigation of the underlyingmechanisms revealed an essential requirement for PI3K-mediated signals during expansion of BL-CFCs. Furthermore,we define a role for PI3K-dependent signalling duringexpansion of haemopoietic progenitors and generation ofmature erythroid and myeloid lineages. These results,summarised in Fig. 7, support a role for PI3K-dependentsignalling at multiple stages of developmental haemopoiesis.

Our data demonstrate a clear decrease in cellularity of EBsgenerated either in the presence of LY294002 or from PDK1-deficient ES cells. In our hands, this does not appear to be dueto increased apoptosis (data not shown), which contrasts to thereport that inhibition of PI3Ks leads to apoptosis in murineblastocysts and of trophoblast stem cells (Riley et al., 2006).However, it is worth noting that we used LY294002 at only 5�M, compared with the 250 �M used by Riley et al. (Riley etal., 2006). It has been reported that LY294002 can also inhibitmTORC1 in vitro (Knight et al., 2006), although this occurs athigher doses of LY294002 than those required to inhibit classIA PI3Ks. Although we cannot formally discount the possibilitythat the more dramatic effects on EB cellularity we observe

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using 10 �M LY294002 are related to effects on mTORC1, webelieve that the remarkably similar results obtained with EScells treated with 5 �M LY294002 and PDK1-null ES cells areconsistent 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 ofmesodermal cells is not drastically altered and temporalpatterns of gene expression are similar. Therefore, we believeour data are most consistent with PI3Ks being required foroptimal proliferation of EB cells. The regulation ofundifferentiated ES cell proliferation via PI3K-dependentsignalling has been reported (Hallmann et al., 2003; Jirmanovaet al., 2002; Paling et al., 2004; Sun et al., 1999; Takahashi et

al., 2003) but very little is known about the involvement ofPI3K signalling pathways during early stages of ES celldifferentiation. Interestingly, S6 phosphorylation is increasedin 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 ascells differentiate. The decrease in proliferation we propose toaccount for our observations is consistent with theconsequences of PDK1 deficiency on embryonic mousedevelopment, where PDK1 has been shown to be important forregulating growth (Lawlor et al., 2002). Similarly, deficiencyof the p110� catalytic isoform of class IA PI3Ks leads to a veryearly embryonic lethality, with very poor growth of p110�-deficient blastocysts reported (Bi et al., 2002). Despite the factthat embryos as early as the one-cell stage express multipleclass IA isoforms (Lu et al., 2004; Riley et al., 2005), thisevidence suggests a non-redundant role for class IA PI3Ks. EScells are derived from the inner cell masses of day 3.5 mouseblastocysts and development of EBs models the earliest stagesof embryonic development. Thus, the effects we observe onEB development are consistent with a requirement for PI3Ksignalling during these early stages.

We were surprised to find that, despite the decrease in EBcellularity, the timing of early haemopoietic differentiationevents – as evidenced by cell surface expression, geneexpression analyses and formation of BL-CFCs – did notappear to be inhibited under conditions of PI3K inhibition orPDK1-deficiency, suggesting that PI3K signalling isdispensable for commitment towards the haemopoietic lineageduring EB development. In fact, we observed an increase inthe proportion of Flk-1+-Kit+ double-positive cells in day 6

Fig. 6. PI3K-dependent signalling is required for optimaldevelopment of erythroid and myeloid lineages. (A) Day 3.75 EB-derived cells generated in the absence of inhibitor were plated intoblast colony culture conditions in the absence (EB 0LY/blast 0LY) orpresence (EB 0LY/blast 5LY) of 5 �M LY294002. In addition, cellsfrom day 3.75 EBs formed in the presence of 5 �M LY294002 wereplated under blast culture conditions without inhibitor (EB 5LY/blast0 LY). After 4 days, blast-colony-derived cells were harvested andidentical numbers of cells for each condition plated into HCAs (inthe absence of LY294002). Numbers of each type of haemopoieticcolony generated are shown. Data represent the average of triplicatecounts (± s.d.) and are representative of three individual experiments.***P<0.005 (B) Day 6 EB-derived cells, differentiated in theabsence of LY294002, were plated into HCAs in the absence (EB0LY/HCA 0LY) or presence of 5 �M LY294002 (EB 0LY/HCA5LY). Data represents the mean (±s.e.m.) from two independentexperiments (n=3-5). Examples showing the size differential ofEry/D colonies that developed in the absence or presence ofLY294002 are shown in the inserts; original magnification 100�(10�/0.4 objective lens was used). Bars, 200 �m. These results arerepresentative 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 �M LY294002 (5 �M LY or 10 �MLY, respectively). Data represent the mean (± s.e.m.) of threeindependent experiments. Examples of overall colony formation aredepicted above the appropriate bar graph; original magnification20� (10�/0.4 objective lens). Bars, 1 mm. In addition, colonymorphologies are shown in the inserts; original magnification 100�(10�/0.4 objective lens). Bars, 200 �m. ***P<0.005 for GMcolonies compared with 0 LY.

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EBs formed in the presence of LY294002. This Flk-1+-Kit+

double-positive population has been proposed to representearly haemopoietic progenitors (Lacaud et al., 2004; Willey etal., 2006), which correlates with our observed increase inhaemopoietic-colony-forming potential of EB-derived cellsgenerated in the presence of PI3K inhibition. Intriguingly, weappear to be detecting a distinct Flk-1high-Kit+ population incells 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 thisdistinct population that has haemopoietic potential. How canwe explain the enhanced presence of cells with haemopoieticpotential in EBs derived in the presence of PI3K inhibition?One possible explanation is that other cell types within thedeveloping EB are differentially more sensitive to the growthinhibitory effects arising from PI3K inhibition, leading to asmall increase in the total proportion of cells that havehaemopoietic potential within the EB. This is certainlysupported by our finding that PDK1-deficient ES cells alsoexhibit enhanced levels of a Flk-1+-Kit+ double-positive cellpopulation. However, we cannot formally exclude thepossibility that our results reflect either the inability ofLY294002 to penetrate to the core of the developing EB, wherethe haemopoietic progenitors are likely to arise, or that anincrease in the subset of cells capable of haemopoieticdifferentiation arises as a direct effect of PI3K inhibition duringearly development of these lineages.

The inability of PDK1-deficient EB-derived progeny toform terminally differentiated haemopoietic cells indicates alikely requirement for PDK1-mediated signals duringdevelopmental haemopoiesis. Our studies reveal a crucialrequirement for PI3K signals during the expansion of theearliest haemopoietic progenitor, the BL-CFC, to form blast

Journal of Cell Science 120 (10)

colonies. Quite remarkably, temporal expression of markergenes was not significantly altered, indicating thatdifferentiation still occurs when PI3Ks are inhibited or PDK1is absent. One caveat to these studies is that we have examinedthe behaviour of only a single PDK1-null ES-cell line, becausean independently derived line was not available. However, thedata generated with the PDK1-deficient cell line supports datagenerated with the PI3K inhibitor LY294002 and, together, areconsistent with PI3K-mediated signals being required forproliferation of haemopoietic progenitors during blast colonyexpansion. This begins to shed light on the signals requiredfor expansion and differentiation of the BL-CFC, about whichlittle is known. VEGF, the ligand for Flk-1, plays a role inblast colony formation, although it is not known whether thisinvolves PI3K signalling. However, PI3K signalling has beenimplicated in VEGF-mediated survival of murine bonemarrow progenitors (Larrivee et al., 2003) and PI3Ks areactivated by VEGF in adult human endothelial cells (Dayaniret al., 2001), so it will be interesting to examine this in thefuture. Consistent with a requirement for class IA PI3K-mediated signals in the expansion of haemopoieticprogenitors, Kit+ haemopoietic progenitors derived fromp85�–/– (Haneline et al., 2006; Huddleston et al., 2003) orp85�–/– p85�–/+ (Haneline et al., 2006; Huddleston et al.,2003) foetal liver show decreased proliferation in response toSCF.

We have also demonstrated a role for PI3K-dependentsignalling in the generation of committed haemopoieticprecursors. Several studies have suggested a role for PI3Ksignalling during foetal liver erythropoiesis (Huddleston et al.,2003; Zhao et al., 2006) and recent work has demonstrated arequirement for the PI3K/AKT signalling pathway in thematuration of foetal liver erythroid progenitors (Ghaffari et al.,2006). Our data are consistent with a requirement for PI3Ksignalling for expansion of haemopoietic progenitors into themature erythroid lineage. In addition, we demonstrate thatinhibition of PI3Ks only during expansion of committedprogenitors dramatically reduced the number of myeloidcolonies formed from haemopoietic progenitors derived fromboth EBs and mouse bone marrow. Although there have beenreports of many cytokines activating PI3Ks (Gold et al., 1994)and of PI3Ks being involved in regulation of proliferation ofmature myeloid lineages (Ali et al., 2004; Fox et al., 2005;Mungalavadla et al., 2005), there is little data available on therole of PI3Ks in development of the myeloid lineage. Duringthe preparation of this manuscript, a requirement for class IAPI3Ks in the function of foetal liver-derived haemopoietic stemcells was reported (Haneline et al., 2006), which demonstratedsignificantly reduced myeloid repopulating activity of p85�–/–

/ p85�–/+ adult HSCs. These studies, together with our data,are consistent with PI3Ks contributing to differentiation of themyeloid lineage.

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

BL-CFC

ES

EB day 3.75 day 6 HCA

Blast Colony

Myeloid

Myeloid

EryP

PI3Ks required for proliferation

PI3Ks required for differentiation

Hp

(Hp)

EryD

EryP

EryD

PDK1 -/-

Fig. 7. PI3K-dependent signalling is required at multiple stages ofdevelopmental haemopoiesis. Our studies have demonstrated thatPI3K signalling is required for proliferation of cells within thedeveloping EB at the early stages of development. However,inhibition of PI3K/PDK1-mediated signals does not blockdifferentiation towards the BL-CFC, as indicated by Flk-1 andbrachurury expression and blast colony formation. A crucialrequirement for PI3Ks and PDK1 was defined during the expansionof the BL-CFC to form blast colonies and this appears to be mainlyat the level of proliferation. Inhibition of PI3Ks only during theHCAs indicates PI3Ks are required for optimal generation ofmyeloid lineages and optimal proliferation of erythroid cells.

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1761PI3Ks regulate developmental haemopoiesis

Materials and MethodsEmbryonic stem cell growth and differentiationThe 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 routinelycultured in knock-out Dulbecco’s modified Eagle’s medium (Invitrogen)supplemented with 103 units/ml murine LIF (ESGRO; Chemicon) as describedpreviously (Paling and Welham, 2005; Paling et al., 2004). Formation of embryoidbodies (EBs) was adapted from Kennedy et al. (Kennedy et al., 1997). ES cells weretrypsinized 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 �g/ml L-ascorbic acid, and 100 �g/ml insulin in Iscove’s modifiedDulbecco’s medium (IMDM, Invitrogen) supplemented with 10 ng/ml basicfibroblast 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 �M LY294002 (Calbiochem).

Colony assaysEBs were harvested, washed in PBS (Invitrogen) and trypsinized for 3 minutes at37°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 �g/ml L-ascorbicacid in IMDM supplemented with 5 ng/ml vascular endothelial growth factor(VEGF; PeproTech), 5 ng/ml IL-6 (Peprotech) and 25% D4T endothelial cellconditioned medium (Kennedy et al., 1997). Colonies were counted following 4days in culture. For the growth of haemopoietic precursors, day 6 EB-derived cellswere plated at 5-10�104 cells/ml in Metho-cult (StemCell Technologies) containing1% (w/v) methylcellulose 15% (v/v) FBS, 1% (w/v) bovine serum albumin (BSA),10 �g/ml insulin 0.2 mg/ml transferrin, 1�10-4 M 2-mercaptoethanol and 2 mMglutamine, 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 clearedby 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 andappeared as very small red clusters of 10-20 erythroblasts. All other colonies werecounted following 8-10 days in culture. Colony images were visualised using anOlympus (Tokyo, Japan) XI51 inverted microscope and objective lenses (aperturesand magnifications are specified in each figure legend) and captured using anOlympus Camedia C4040 digital camera. Data were analysed for statisticalsignificance using two-tailed paired Student’s t-tests.

Cell lysates and immunoblottingEBs were washed three times in PBS and lysed directly in ice-cold solubilisationbuffer, as described previously (Welham et al., 1994). Insoluble material wasremoved by centrifugation at full speed for 2 minutes in an Eppendorfmicrocentrifuge at 4°C. Protein concentrations were determined using the Bio-Rad(Hercules, CA) protein assay kit according to the manufacturer’s instructions and10 �g of each cell lysate separated by SDS-PAGE an transferred onto nitrocellulose(Welham et al., 1994). Immunobloting was performed using primary antibodies at1:1000 dilution: rabbit polyclonal antibodies recognising double phosphorylatedribosomal protein S6 at Ser235-Ser236 (anti-P-S6, Cell Signal Technologies, 2211)or SHP-2 (Santa Cruz Biotechnology) or goat polyclonal antibody againstglyceraldehyde-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 describedpreviously (Welham et al., 1994).

Flow cytometryEBs were trypsinised and the cells resuspended in ice-cold wash buffer [PBScontaining 2% (v/v) FBS and 0.1% (w/v) sodium azide]. Non-specific binding toFc receptors was blocked by incubation with mouse anti-CD16/CD32 anibody(Fc�III/II receptors; 1:2500; BD PharMingen) for 30 minutes on ice. Cells werethen stained on ice for a further 30 minutes with phycoerythrin (PE)-conjugatedanti-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 bufferand incubated with (PE-Cy5)-conjugated streptavidin (1:100; DAKO) for a further30 minutes. Flow cytometry was performed using a FACSCanto cytometer (BectonDickenson) and the data were analysed with FACSDiva software. Dead cells wereexcluded from analyses based on forward and side scatter parameters.

Gene expression analysesEBs were collected, washed in PBS and RNA was purified using TRIzol reagent(Invitrogen), following the manufacturer’s instructions. All RNA samples weretreated with DNase I (Promega) before cDNA synthesis to eliminate anycontaminating genomic DNA. RNA (1 �g) was reverse transcribed into cDNA with

Oligo(dT)15 (Promega) using SuperScript II (Invitrogen). Gene-specific PCR wascarried 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-TGTC -TCTCCAGTTTCCCTGC-3; Kit antisense, 5-TTCAGGGACTCATGGGCTCA-3; flk1 sense, 5-CACCTGGCACTCTCCACCTTC-3; flk1 antisense, 5-GATT -TCATCCCACTACCGAAAG-3; scl sense, 5-ATTGCACACACGGGATTCTG-3; scl antisense, 5-GAATTCAGGGTCTTCCTTAG-3; gata1 sense, 5-ACTC -GTCATACCACTAAG-3; gata1 antisense, 5-AGTGTCTGTAGGCCTCAG-3;�-major sense, 5-CGATGAAGTTGGTGGTGAGG-3; �-major antisense, 5-CACAATCACGATCATATTGCC-3; �-H1 sense, 5-AGTCCCCATGGAG TCA -AAGA-3; �-H1 antisense, 5-CTCAAGGAGACCTTTGCTCA-3; gapdh sense,5-TGGAGCCAAACGGGTCATC-3; gapdh antisense, 5-GCTCTGGGATGAC -CTTGCC-3; �-actin sense, 5-TAGGCACCAGGGTGTGATGG-3; �-actinantisense, 5-CATGGCTGGGGTGTTGAAGG-3.

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

We thank Helen Wheadon and Nick Paling for initial assistancewith ES differentiation, Dario Alessi for PDK1–/– ES cells, andGeorge Lacaud and Gordon Keller for D4T cell lines. This work wasfunded by the Wellcome Trust (grant reference 070316/Z/03/Z).

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