Essential role of PDK1 in regulating cell size and development in mice
2002; Springer Nature; Volume: 21; Issue: 14 Linguagem: Inglês
10.1093/emboj/cdf387
ISSN1460-2075
AutoresMargaret A. Lawlor, Alfonso Mora, Peter R. Ashby, Michayla R. Williams, Victoria Murray-Tait, Lorraine Malone, Alan R. Prescott, John M. Lucocq, Dario R. Alessi,
Tópico(s)Nuclear Structure and Function
ResumoArticle15 July 2002free access Essential role of PDK1 in regulating cell size and development in mice Margaret A. Lawlor Corresponding Author Margaret A. Lawlor MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Alfonso Mora Alfonso Mora MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Peter R. Ashby Peter R. Ashby Cell and Developmental Biology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Michayla R. Williams Michayla R. Williams MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Victoria Murray-Tait Victoria Murray-Tait Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Lorraine Malone Lorraine Malone Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Alan R. Prescott Alan R. Prescott Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author John M. Lucocq John M. Lucocq Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Dario R. Alessi Dario R. Alessi MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Margaret A. Lawlor Corresponding Author Margaret A. Lawlor MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Alfonso Mora Alfonso Mora MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Peter R. Ashby Peter R. Ashby Cell and Developmental Biology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Michayla R. Williams Michayla R. Williams MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Victoria Murray-Tait Victoria Murray-Tait Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Lorraine Malone Lorraine Malone Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Alan R. Prescott Alan R. Prescott Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author John M. Lucocq John M. Lucocq Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Dario R. Alessi Dario R. Alessi MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK Search for more papers by this author Author Information Margaret A. Lawlor 1, Alfonso Mora1, Peter R. Ashby2, Michayla R. Williams1, Victoria Murray-Tait3, Lorraine Malone3, Alan R. Prescott3, John M. Lucocq3 and Dario R. Alessi1 1MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK 2Cell and Developmental Biology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK 3Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3728-3738https://doi.org/10.1093/emboj/cdf387 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info PDK1 functions as a master kinase, phosphorylating and activating PKB/Akt, S6K and RSK. To learn more about the roles of PDK1, we generated mice that either lack PDK1 or possess PDK1 hypomorphic alleles, expressing only ∼10% of the normal level of PDK1. PDK1−/− embryos die at embryonic day 9.5, displaying multiple abnormalities including lack of somites, forebrain and neural crest derived tissues; however, development of hind- and midbrain proceed relatively normally. In contrast, hypomorphic PDK1 mice are viable and fertile, and insulin injection induces the normal activation of PKB, S6K and RSK. Nevertheless, these mice are 40–50% smaller than control animals. The organ volumes from the PDK1 hypomorphic mice are reduced proportionately. We also establish that the volume of a number of PDK1-deficient cells is reduced by 35–60%, and show that PDK1 deficiency does not affect cell number, nuclear size or proliferation. We provide genetic evidence that PDK1 is essential for mouse embryonic development, and regulates cell size independently of cell number or proliferation, as well as insulin's ability to activate PKB, S6K and RSK. Introduction The 3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a key role in regulating the activity of a group of insulin and growth factor-stimulated protein kinases that belong to the AGC subfamily of protein kinases (Niederberger and Schweingruber, 1999; Toker and Newton, 2000; Alessi, 2001). These include isoforms of protein kinase B (PKB, also known as Akt) (Brazil and Hemmings, 2001; Scheid and Woodgett, 2001), p70 ribosomal S6 kinase (S6K) (Avruch et al., 2001; Volarevic and Thomas, 2001) and p90 ribosomal S6 kinase (Frodin et al., 2000). Once activated, these enzymes mediate many of the diverse effects of insulin and growth factors on cells by phosphorylating key regulatory proteins that play important roles controlling processes such as cell survival, proliferation, nutrient uptake and storage. There is only a single gene encoding PDK1 in mammals. PDK1 functions to activate AGC kinase members by phosphorylating these enzymes at a conserved residue known as the T-loop residue, which is located in the core of their kinase catalytic domain (Niederberger and Schweingruber, 1999; Toker and Newton, 2000; Alessi, 2001). The activation of PKB and S6K by PDK1 is dependent on prior activation of the phosphoinositide 3-kinase (PI 3-kinase). This produces the second messenger, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], which binds to the pleckstrin homology domains of PKB and PDK1, recruiting these enzymes to the plasma membrane where PKB is phosphorylated by PDK1 (Brazil and Hemmings, 2001; Scheid and Woodgett, 2001). In contrast, S6K does not interact with PtdIns(3,4,5)P3 but, instead, PtdIns(3,4,5)P3 stimulates the phosphorylation of a Thr residue located C-terminal to the S6K catalytic domain in a motif known as the hydrophobic motif, by an as yet unknown mechanism (Avruch et al., 2001; Volarevic and Thomas, 2001). The phosphorylation of S6K at this site enhances the ability of PDK1 to interact with, phosphorylate and activate S6K (Biondi et al., 2001). The activation of RSK isoforms is initiated by ERK/MAP kinase phosphorylation in response to agonists that activate the Ras/Raf pathway. In order for RSK to be activated by ERK, RSK also needs to be phosphorylated at the T-loop residue of its N-terminal kinase domain by PDK1 (Frodin and Gammeltoft, 1999). The key role that PDK1 plays in activating certain AGC kinase members was established by the finding that mouse embryonic stem (ES) cells lacking PDK1 failed to activate PKB, S6K and RSK in response to stimuli that trigger the activation of these enzymes in wild-type ES cells (Williams et al., 2000). It was perhaps unexpected that ES cells lacking PDK1 were viable, because PKB and RSK have been reported to play important roles in regulating survival and proliferation in other cell types (Nebreda and Gavin, 1999; Lawlor and Alessi, 2001). Furthermore, knocking out PDK1 homologues in Saccharomyces cerevisiae (Casamayor et al., 1999; Inagaki et al., 1999), Schizosaccharomyces pombe (Niederberger and Schweingruber, 1999), Caenorhabditis elegans (Paradis et al., 1999) and Drosophila (Cho,K.S. et al., 2001; Rintelen et al., 2001) have revealed that PDK1 is required for the normal development and viability of these organisms. To learn more about the roles that PDK1 plays in mammals, we generated and analysed the phenotype of both PDK1 knockout mice and PDK1 hypomorphic mutant mice that express markedly lower levels of PDK1 in all tissues examined. Results Manipulating levels of PDK1 in ES cells We generated mouse ES cell lines called PDK1fl/fl, in which the neomycin resistance gene flanked with the loxP CRE excision sites was inserted in an intron sequence between exons 2 and 3 of both alleles of the PDK1 gene (Figure 1A). We observed that these cells were hypomorphic for PDK1 expression, as they had markedly lower levels of PDK1 protein (Figure 1B) and PDK1 kinase activity (Figure 1C) compared with the control PDK1+/+ ES cells, while the levels of PKBα protein were identical in both cell types. Consistent with the presence of the neomycin resistance gene causing the reduction in PDK1 levels, its removal using the CRE recombinase, to generate PDK1flΔneo/flΔneo ES cells, resulted in restoration of PDK1 protein and kinase activity levels to those found in PDK1+/+ ES cells. The heterozygote PDK1+/fl ES cells possessed a normal level of PDK1 protein and kinase activity (Figure 1B and C). The PDK1+/−, PDK1+/fl and PDK1+/flΔneo ES cells were injected into murine blastocysts to generate mice possessing these genotypes using standard procedures. Figure 1.Generation of hypomorphic PDK1 ES cells. (A) Diagram illustrating the 5′ end of the PDK1 gene and the different alleles that we have generated. The black boxes represent exons, continuous lines represent introns, triangles represent loxP sites and the neo box represents a neomycin resistance gene cassette. The positions of the PCR primers used to genotype the ES cells and mice are indicated with arrows. +, wild-type allelle; fl, allelle containing the neomycin resistance gene flanked with loxP sites between exons 2 and 3 as well as a loxP site in intron 4; flΔneo, allelle in which the neomycin resistance gene cassette was excised with the CRE recombinase but still possesses a loxP sites flanking exons 3 and 4; −, denotes the allele in which exon 3 and 4 have been removed with CRE and this also results in a frame-shift mutation (Williams et al., 2000), which would ablate expression of the PDK1 protein beyond exon 2, which includes the kinase and pleckstrin homology domain. (B) The indicated ES cell lines were grown to confluence, lysed and 20 μg of protein extract was electrophoresed on a 10% SDS–polyacrylamide gel and immunoblotted with antibodies that recognize murine PDK1 (top panel) or PKBα (bottom panel). Similar results were obtained in three separate experiments. (C) The ES cells were lysed, and PDK1 immunoprecipitated and assayed. Data are presented as the mean of two separate experiments ± SEM with each determination carried out in triplicate. Download figure Download PowerPoint Embryonic lethality of PDK1−/− mice The PDK1+/− heterozygous mice were healthy and displayed no obvious phenotypes. In an attempt to generate complete PDK1 knockout mice, matings were set up between heterozygous PDK1+/− mice and the resulting progeny were genotyped (Table I). No PDK1−/− postnatal mice were ever recovered, indicating that this genotype resulted in an embryonic lethal phenotype. We next analysed the genotype of embryos from days 7.0 to 12.5 derived from PDK1+/− matings, which revealed the presence of PDK1−/− embryos at the expected Mendelian distribution at embryonic day (E) 7.5 to E9.5 (Table I). We were not able to isolate PDK1−/− embryos at E10 or later, indicating that the mutant embryos died and were reabsorbed after E9.5. Table 1. Mice matings reported in this study Cross Stage Genotype Total PDK1+/− × PDK1+/− E7.5 PDK1+/+ (25%) PDK1+/− (45%) PDK1−/− (30%) 20 PDK1+/− × PDK1+/− E8.5 PDK1+/+ (27%) PDK1+/− (50%) PDK1−/− (23%) 26 PDK1+/− × PDK1+/− E9.5 PDK1+/+ (23%) PDK1+/− (52%) PDK1−/− (25%) 61 PDK1+/− × PDK1+/− Weaning PDK1+/+ (34%) PDK1+/− (66%) PDK1−/− (0%) 81 PDK1+/fl × PDK1+/fl Weaning PDK1+/+ (32%) PDK1+/fl (47%) PDK1fl/fl (21%) 106 PDK1fl/fl × PDK1+/− Weaning PDK1+/fl (63%) PDK1−/fl (37%) 78 PDK1fl/fl × PDK1fl/fl Weaning PDK1fl/fl (100%) 27 PDK1−/fl × PDK1−/fl Weaning PDK1−/fl (30%) PDK1fl/fl (70%) PDK1−/− (0%) 20 The indicated matings were set up and the progeny genotyped as described in Materials and methods. The percentage of each genotype observed is indicated in parenthesis. Total indicates the number of progeny analysed. Mice were weaned as described in Materials and methods. Therefore, to examine the pattern of PDK1 mRNA expression in E7–9 embryos, we performed whole-mount in situ hybridization (Figure 2). This analysis revealed that PDK1 mRNA was expressed ubiquitously in all embryonic and extra-embryonic tissues from E7 to E9. Figure 2.PDK1 is ubiquitously expressed in early embryos. Whole-mount mRNA in situ hybridization for PDK1 expression was carried out on PDK1+/+ embryos at the indicated developmental stages. The embryos were processed in the presence (+) or absence (−) of PDK1 probe. Similar results were observed in two separate experiments. Download figure Download PowerPoint Analysis of PDK1−/− phenotype At E7.0, PDK1−/− embryos are already smaller than their PDK1+/+ littermates (Figure 3A). They have defined embryonic and extra-embryonic segments, but no obvious external phenotypic difference. By E8.0 (four to six somites), PDK1−/− embryos are significantly smaller than their PDK1+/+ littermates in length, possess very small headfolds, display no visible somites or posterior mesoderm, and have a small allantois. Extra-embryonic membranes are present in the PDK1−/− embryos. It is not clear whether these embryos have specific phenotype or are retarded in development. Figure 3.Analysis of PDK1−/− and PDK1+/+ embryos. (A–E) PDK1−/− and PDK1+/+ embryos were dissected in PBS and imaged on a Leica M275 microscope and whole-mount photographs were taken. hd, head; hf, headfolds; he, heart; al, allantois; eme, extra-embryonic membranes; psm, presomitic mesoderm; exr, extra-embryonic region; emr, embryonic region; nt, neural tube; so, somites. (F) A representative image of E7.5 PDK1+/+ and PDK1−/− embryonic endoderm cells stained with the lipid dye DilC16(3) using a Zeiss LSM410 microscope. (G) The size of the E7.5 PDK1+/+ and PDK1−/− embryonic endoderm cells was quantitated as described in Materials and methods. Two embryos of each genotype were analysed, with 120 cells in each embryo being measured (P < 0.0004). Download figure Download PowerPoint However, at E8.5 (eight to 10 somites) the phenotypic differences in the PDK1−/− embryos become obvious. The head region is less developed, resembling a small compact mass, there are still no somites, although a presomitic mesoderm is present, and a forming neural tube is visible as is an enlarging allantois (Figure 3C). At E9.0–9.5, the PDK1+/+ embryos have turned and are floating inside their amniotic sacs with developed umbilical vessels. In contrast, the PDK1−/− embryos have not turned and lie along the top edge of their amniotic sacs with their heads indenting the membranes and interrupting the normal smooth outline (data not shown). The allantois of the E9.0 PDK1−/− embryos have developed further and contact the placenta and, although no vascular differentiation is seen (Figure 3D and E), blood vessels are visible in embryos and yolk sac (data not shown). We also investigated the size of the E7.5 PDK1−/− and PDK1+/+ embryonic endoderm cells using quantitative stereological procedures (see Materials and methods). This analysis revealed that PDK1−/− embryonic endoderm cells were markedly smaller, being only 40% of the size of wild-type cells (Figure 3F and G). To examine the phenotype of the E9–9.5 PDK1−/− embryos more closely, embryos were examined by scanning electron microscopy (Figure 4). This analysis revealed features of both advanced and abnormal development: the posterior growth of the PDK1−/− embryos seems to have halted; the somites are absent and the presomitic mesoderm appears swollen, which may be due to accumulated somitic cells (Figures 3E and 4A); the neural tube has closed, but the dorsal root ganglia are absent (Figure 4A and C); in some embryos an otic pit is clearly visible (Figure 4D), but no branchial arches are seen (Figure 4G and H); and no heart or heart tube is present (Figure 4G). Figure 4.Scanning electron microscope images of E8.5 PDK1+/+ and E9.5 PDK1−/− embryos. Embryos were dissected into PBS and prepared for the scanning electron microscope as described in Materials and methods. (A and C–E) Dorsal views of E9.5 PDK1−/− embryos; (F) and (H) are dorsal and ventral view of a E9.5 PDK1+/+ embryos, respectively. (G) A ventral view of a mutant embryo, whereas (B) is a dorsal view of an E8.5 PDK1+/+ embryo. hd, head; hf, head folds; al, allantois; nt, neural tube; oph, open portion of the hindbrain; pos, pre-otic sulcus; psm, presomitic mesoderm; rp, Rathke's pouch; op, otic pit; so, somites; ba, branchial arches; fb, forebrain; *, no somites; #, no dorsal root ganglia; §, no heart. Download figure Download PowerPoint Notably, the head has developed normally to some extent in E9.5 PDK1−/− embryos (Figure 4D–H). The headfolds seen at earlier stages (Figure 3B) are fused, and there is a marked pre-otic sulcus and the beginnings of the open portion of the hindbrain (Figure 4E). Ventrally, the absence of the forebrain, including the eyes, is obvious (Figure 4G), a visible pit is present with the correct position and timing to be Rathke's pouch, invaginating towards the diencephalon to form the pituitary gland, which is diagnostic of a normal E9.5 embryo (Figure 4G and H). There were no detectable abnormalities in PDK1+/− embryos, which were indistinguishable from the PDK1+/+ embryos. Analysis of hypomorphic PDK1fl/fl and PDK1−/fl mutant mice As it was not possible to generate viable PDK1−/− mice, we decided to breed PDK1fl/fl and PDK−/fl mice to determine whether the tissues of these mice showed reduced levels of PDK1 and whether they displayed any discernable phenotype. The PDK1fl/fl mice were viable and born at a marginally (∼5%) lower than expected Mendelian frequency (Table I). Viable PDK1−/fl mice were also obtained, however, which were born at an ∼25% lower frequency than predicted (Table I). Both the PDK1fl/fl and the PDK1−/fl mice were fertile (Table I). In Figure 5, we quantify the specific activities of PDK1 in eight different tissues of 4-month-old male PDK1+/+, PDK1+/−, PDK1+/fl, PDK1fl/fl, PDK1−/fl and PDK1flΔneo/flΔneo mice. In all tissues investigated, PDK1fl/fl mice had 3- to 5-fold lower PDK1 kinase activities compared with PDK1+/+ mice, whereas PDK1−/fl mice had 5- to 10-fold lower PDK1 kinase activity. As expected, the PDK1 kinase activity was restored to the levels observed in wild-type animals in PDK1flΔneo/flΔneo mice in which the neomycin resistance gene had been excised. In most tissues, the PDK1+/− and PDK1+/fl mice possessed a 25–50% reduction in PDK1 activity relative to wild-type animals (Figure 5). Figure 5.Reduced PDK1 activity in PDK1fl/fl and PDK1−/fl mice. Tissue extracts from the indicated mice were prepared. PDK1 was immunoprecipitated and assayed. Data are presented as the mean ± SEM and is representative of three separate experiments with each determination carried out in triplicate. Δneo, flΔneo/flΔneo. Download figure Download PowerPoint Normal activation of PKB, S6K and RSK in hypomorphic PDK1−/fl mice To determine whether reduced expression of PDK1 in PDK1−/fl mice impaired activation of characterized PDK1 substrates, PDK1+/fl and PDK1−/fl littermates were injected with varying doses of insulin, extracts generated from skeletal muscle, liver and adipose tissue, and PKB, S6K and RSK activity was assessed (Figure 6). Activation of PKB was determined by immunoblotting cell lysates with PKB phospho-specific antibodies recognizing the T-loop PDK1 phosphorylation site (Thr308 in PKBα), as well as the hydrophobic motif (Ser473 in PKBα). There was no major difference at any of the doses of insulin used to stimulate the phosphorylation of PKB at its T-loop or hydrophobic motif in the tissues examined (Figure 6A). Even at the lowest dose of insulin that induced a detectable phosphorylation of PKB in the PDK1+/fl mice, a similar phosphorylation was observed in the tissues of the PDK1−/fl mice. S6K1 activity was examined by immunoprecipitating this enzyme and measuring its ability to phosphorylate a peptide substrate (Figure 6B). The dose of insulin employed was the lowest amount that gave a significant activation of S6K1 in PDK1+/+ mice (data not shown). These experiments revealed that S6K1 was activated similarly in PDK1+/fl and PDK1−/fl mice. Insulin also stimulated ERK1 phosphorylation in skeletal muscle (Figure 6C), but not detectably in liver or adipocytes (data not shown). This enabled us to compare RSK activation in the skeletal muscle of PDK1+/fl and PDK1−/fl mice. RSK was activated similarly by insulin in both PDK1+/fl and PDK1−/fl mice (Figure 6C). We also investigated the activity of PKB, S6K and RSK isoforms in non-fasted PDK1+/fl and PDK1−/fl mice not injected with insulin. In these animals, PKB, S6K and RSK had similar basal activity in the muscle, liver and adipose tissues to the levels observed in fasted mice not injected with insulin (data not shown). Figure 6.Activation of PKB, S6K1 and RSK is normal in PDK1−/fl mice. (A) Mice were fasted overnight and injected with either saline (−) or increasing doses of insulin namely (0.0625, 0.625, 12.5 or 250 mU/g of mouse) for 10 min and the indicated tissues harvested as described in Materials and methods. Lysates were immunoblotted with antibodies that recognize PKBα phosphorylated at Thr308 (top panel, P-T308) and Ser473 (middle panel, P-S473) or the PKBα protein (bottom panel, Total PKB). (B) As above, except that the mice were injected with saline or insulin (12.5 mU/g of mouse) for 20 min. S6K1 was immunoprecipitated and assayed. (C) As in (B) except that cell lysates from skeletal muscle were either immunoblotted with antibodies recognizing phosphorylated ERK1/ERK2 (P-ERK1) or the ERK1/ERK2 proteins (Total ERK1), as well as RSK1 and RSK2 isoforms. It should be noted that the levels of ERK2 in skeletal muscle appear to be lower than we can detect. RSK isoforms were also immunoprecipitated and assayed. For the immunoblotting data shown, results were obtained in two separate experiments. For the S6K and RSK enzymic assays, the data are presented as the mean ± SEM of two separate experiments with each determination carried out in triplicate. Download figure Download PowerPoint Small size of PDK1 hypomorphic mice Both male and female hypomorphic PDK1fl/fl mice were ∼30% smaller from birth than their PDK1+/+ or PDK1+/fl littermates, and remained smaller throughout their adult life. In the case of the male mice, the difference in weight between the PDK1fl/fl and PDK1+/+ mice increased to ∼45% by 6 months of age, whereas the difference between the female PDK1fl/fl and PDK1+/+ mice remained ∼30% (Figure 7A). There was an even more marked difference in weight between the PDK1−/fl and PDK1+/fl littermates. The male and female PDK1−/fl mice were 45–50% and ∼35%, respectively, smaller than their PDK1+/+ littermates (Figure 7B and C). Consistent with the decrease in size of the PDK1fl/fl and PDK1−/fl mice being due to reduced levels of PDK1, both male and female PDK1flΔneo/flΔneo mice were similar in size to the PDK1+/+ and PDK1+/fl mice (Figure 7C). Figure 7.Reduced size of PDK1fl/fl and PDK1−/fl mice. (A) The mean body weight of male and female PDK1+/+, PDK1+/fl and PDK1fl/fl mice at the indicated age. Values represent the mean ± SEM for each data point. The numbers (n) of each genotype are indicated on the graph. (B) A representative photograph of PDK1−/fl and PDK1+/fl littermates at E14.5, 1 week and 6 weeks of age is shown. (C) As in (A) for the indicated mice genotypes. (D) The organ volume of kidney, pancreas, spleen and adrenal gland of PDK1+/fl and PDK1−/fl littermates was measured using the Cavalieri method as described in Materials and methods. The data are presented as the mean ± SEM of three different mice per genotype, and only error bars larger than the size of the symbols are shown. Download figure Download PowerPoint We next compared organ volumes of the kidney, pancreas, spleen and adrenal gland from PDK1+/fl and PDK1−/fl littermates using sections analysed by the quantitative Cavalieri method (Gundersen and Jensen, 1987). These four organs had an average volume that was ∼50% smaller in PDK1−/fl mice compared with their PDK1+/fl littermates (Figure 7D). Evidence that PDK1 regulates cell size The reduced size of organs in the PDK1 hypomorphic mice could be explained if the cells are of normal size but are fewer in number and/or if the cells are smaller and just as numerous. To distinguish between these possibilities, we employed a quantitative unbiased method for determining cell volume in intact organs (as described in Materials and methods). This method uses a stereological approach called the disector principle to sample the cells in an unbiased manner before estimating their volume [other non-stereological methods of volume estimation may yield biased estimates (reviewed in Sterio, 1984; Gundersen, 1986)]. In the zona fasciculata of the adrenal gland, we found that PDK1−/fl cells were 45% smaller than PDK1+/fl cells (Figure 8B), and in Figure 8A, we show representative sections of these adrenal cells. It is important to note that the size of the nuclei in both PDK1−/fl and PDK1+/fl types was not significantly different (Figure 8A and C). Combining data on cell volume with stereological estimates of the aggregate volume of cells as described in Materials and methods, we could estimate the total number of cells in the zona fasciculata. These data showed that there was no significant difference in the number of cells in this region of the adrenal gland of the PDK1+/fl and PDK1−/fl mice (Figure 8D). Figure 8.PDK1−/fl mice possess smaller adrenal cells. (A) The top panel shows a micrograph (magnification ×250) of zona fasciculata cells of the adrenal gland from PDK1−/fl (right) and PDK1+/fl (left) mice. The panels are representative of the micrographs taken. (B) The cell size was measured using the disector principle as described in Materials and methods. The data are presented as the mean ± SEM from three separate experiments as a percentage of the PDK1+/fl cell size. The asterisk denotes that size of PDK1−/fl cells is significantly (P < 0.005) lower than that of PDK1+/fl cells. (C) The size of the nuclei was also measured using the disector principle. The data are presented as the mean ± SEM from two separate experiments as a percentage of the PDK1+/fl nuclear size. (D) The number of cells present in the zona fasciculata was determined by dividing the total volume of zona fasciculata cells by the volume of an individual cell. The data are presented as the mean ± SEM from three separate experiments. Download figure Download PowerPoint Proliferation and size of fibroblasts from PDK1-reduced expression mice Mouse embryonic fibroblasts (MEF) were isolated from the PDK1+/fl and PDK1−/fl embryos and, consistent with other results in this study, the PDK1−/fl cells possessed 6-fold lower PDK1 kinase activity than PDK1+/fl cells (Figure 9A). We measured the size of these cells selected in disectors by the Cavalieri method (Figure 9B), and these results revealed that PDK1−/fl cells were 35% smaller than PDK1+/fl cells. We also compared the proliferation rates of the PDK1+/fl and PDK1−/fl MEF cells over a 5-day period in culture and found that there was no significant difference in the rate at which they multiplied (Figure 9C). We also analysed the viability of PDK1+/fl and PDK1−/fl MEF cells using an apoptosis TUNEL assay. Very low levels of apoptotic cells were observed in PDK1+/fl and PDK1−/fl MEF cells grown in serum, and apoptosis of these cells was induced by the addition of non-specific kinase inhibitor staurosporine. No differences in apoptosis were observed between the PDK1+/fl and PDK1−/fl MEF cells (Figure 9D). Depriving the MEF cells of serum for 24 h did not induce significant increase in basal levels of apoptosis in either cell type (data not shown). Figure 9.PDK1−/fl MEF cells are smaller than PDK1+/fl cells but proliferate at the same rate. (A) PDK1 was immunoprecipitate
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