Members of the Foxo family, Foxo1 (Fkhr), Foxo3 (Fkhrl1), and Foxo4 (Afx), are mammalian homologs of daf-16, which influences life span and energy metabolism in Caenorhabditis elegans. Mammalian FOXO proteins also play important roles in cell cycle arrest, apoptosis, stress resistance, and energy metabolism. In this study, we generated Foxo1-deficient mice to investigate the physiological role of FOXO1. The Foxo1-deficient mice died around embryonic day 11 because of defects in the branchial arches and remarkably impaired vascular development of embryos and yolk sacs. In vitro differentiation of embryonic stem cells demonstrated that endothelial cells derived from wild-type and Foxo1-deficient embryonic stem cells were able to produce comparable numbers of colonies supported by a layer of OP9 stromal cells. Although the morphology of the endothelial cell colonies was identical in both genotypes in the absence of exogenous vascular endothelial growth factor (VEGF), Foxo1-deficient endothelial cells showed a markedly different morphological response compared with wild-type endothelial cells in the presence of exogenous VEGF. These results suggest that Foxo1 is essential to the ability of endothelial cells to respond properly to a high dose of VEGF, thereby playing a critical role in normal vascular development. Members of the Foxo family, Foxo1 (Fkhr), Foxo3 (Fkhrl1), and Foxo4 (Afx), are mammalian homologs of daf-16, which influences life span and energy metabolism in Caenorhabditis elegans. Mammalian FOXO proteins also play important roles in cell cycle arrest, apoptosis, stress resistance, and energy metabolism. In this study, we generated Foxo1-deficient mice to investigate the physiological role of FOXO1. The Foxo1-deficient mice died around embryonic day 11 because of defects in the branchial arches and remarkably impaired vascular development of embryos and yolk sacs. In vitro differentiation of embryonic stem cells demonstrated that endothelial cells derived from wild-type and Foxo1-deficient embryonic stem cells were able to produce comparable numbers of colonies supported by a layer of OP9 stromal cells. Although the morphology of the endothelial cell colonies was identical in both genotypes in the absence of exogenous vascular endothelial growth factor (VEGF), Foxo1-deficient endothelial cells showed a markedly different morphological response compared with wild-type endothelial cells in the presence of exogenous VEGF. These results suggest that Foxo1 is essential to the ability of endothelial cells to respond properly to a high dose of VEGF, thereby playing a critical role in normal vascular development. The Foxo family is one of the forkhead-type transcription factor families and is unique in that its members are downstream components of the insulin signaling pathway involving phosphatidylinositol 3-kinase/Akt (1Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5333) Google Scholar). They are also mammalian homologs of the daf-16 gene in Caenorhabditis elegans, which is essential for the extension of life span and dauer formation in C. elegans. DAF-16 confers resistance to stress such as heat and UV light, and dauer formation is observed under adverse conditions such as food deprivation (2Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1517) Google Scholar, 3Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1187) Google Scholar). Just as in C. elegans, in mammals, the Foxo family functions to regulate the transcription of genes involved in stress resistance and energy metabolism. Genes regulated by FOXO proteins are grouped into four categories. The first group includes the genes for proteins involved in cell cycle arrest and DNA repair such as p27 and GADD45 (4Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404 (R. H.G. J.): 782-787Crossref PubMed Scopus (1207) Google Scholar, 5Tran H.B.A. Grenier J.M. Datta S.R. Fornace Jr., A.J. DiStefano P.S. Chiang L.W. Greenberg M.E. Science. 2002; 296 (H. B.): 530-534Crossref PubMed Scopus (691) Google Scholar, 6Furukawa-Hibi Y. Yoshida-Araki K. Ohta T. Ikeda K. Motoyama N. J. Biol. Chem. 2002; 277: 26729-26732Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 7Dijkers P.F. Medema R.H. Lammers J.W. Koenderman L. Coffer P.J. Curr. Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). The second group is made up of genes whose products are related to apoptosis such as FasL and Bim (1Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5333) Google Scholar, 7Dijkers P.F. Medema R.H. Lammers J.W. Koenderman L. Coffer P.J. Curr. Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). The third group comprises genes for proteins related to resistance to oxidative stress such as manganese-containing superoxide dismutase and catalase (8Kops G.J. Dansen T.B. Polderman P.E. Saarloos I. Wirtz K.W. Coffer P.J. Huang T.T. Bos J.L. Medema R.H. Burgering B.M. Nature. 2002; 419: 316-321Crossref PubMed Scopus (1237) Google Scholar, 9Nemoto S. Finkel T. Science. 2002; 295: 2450-2452Crossref PubMed Scopus (725) Google Scholar). The last group is composed of genes whose products are involved in energy metabolism such as glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, and insulin-like growth factor-1-binding protein-1 (10Nakae J. Kitamura T. Silver D.L. Accili D. J. Clin. Investig. 2001; 108: 1359-1367Crossref PubMed Scopus (485) Google Scholar, 11Barthel A. Schmoll D. Kruger K.D. Bahrenberg G. Walther R. Roth R.A. Joost H.G. Biochem. Biophys. Res. Commun. 2001; 285: 897-902Crossref PubMed Scopus (95) Google Scholar, 12Yeagley D. Guo S. Unterman T. Quinn P.G. J. Biol. Chem. 2001; 276: 33705-33710Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 13Durham S.K. Suwanichkul A. Scheimann A.O. Yee D. Jackson J.G. Barr F.G. Powell D.R. Endocrinology. 1999; 140: 3140-3146Crossref PubMed Scopus (133) Google Scholar). We previously reported that each member of the Foxo family (Foxo1 (Fkhr), Foxo3 (Fkhrl1), and Foxo4 (Afx)) show a tissue-specific and developmentally specific expression pattern (14Furuyama T. Nakazawa T. Nakano I. Mori N. Biochem. J. 2000; 349: 629-634Crossref PubMed Scopus (541) Google Scholar). Foxo4 mRNA is abundant in skeletal muscle throughout life, whereas Foxo1 mRNA is abundantly found in adipose tissues. Foxo3a mRNA is scant during ontogeny, but becomes abundant after birth (14Furuyama T. Nakazawa T. Nakano I. Mori N. Biochem. J. 2000; 349: 629-634Crossref PubMed Scopus (541) Google Scholar). Moreover, recent studies showed that FOXO1 plays important roles in the differentiation of adipocytes and in the myotube fusion of myoblasts in vitro (15Nakae J. Kitamura T. Kitamura Y. Biggs III, W.H. Arden K.C. Accili D. Cavenee W.K. Wright C.V. Dev. Cell. 2003; 4: 119-129Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 16Bois P.R. Grosveld G.C. EMBO J. 2003; 22: 1147-1157Crossref PubMed Scopus (137) Google Scholar). It is therefore suggested that each member plays a distinct role in a tissue-specific and developmentally specific manner in vivo. Indeed, studies using Foxo1 transgenic mice and heterozygous Foxo1-deficient mice showed that FOXO1 is involved in the differentiation of beta-cells in the pancreas and energy metabolism (15Nakae J. Kitamura T. Kitamura Y. Biggs III, W.H. Arden K.C. Accili D. Cavenee W.K. Wright C.V. Dev. Cell. 2003; 4: 119-129Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 17Nakae J. Biggs III, W.H. Kitamura T. Cavenee W.K. Wright C.V. Arden K.C. Accili D. Nat. Genet. 2002; 32: 245-253Crossref PubMed Scopus (526) Google Scholar). In this study, we generated Foxo1-null mice to address the role of FOXO1 in vivo. Foxo1-null mice died at around embryonic day (E) 1The abbreviations used are: E, embryonic day; ES, embryonic stem; VEGF, vascular endothelial growth factor; mAb, monoclonal antibody; VE-cadherin, vascular endothelial cadherin; RT, reverse transcription; PFA, paraformaldehyde; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; PECAM-1, platelet endothelial cell adhesion molecule-1. 1The abbreviations used are: E, embryonic day; ES, embryonic stem; VEGF, vascular endothelial growth factor; mAb, monoclonal antibody; VE-cadherin, vascular endothelial cadherin; RT, reverse transcription; PFA, paraformaldehyde; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; PECAM-1, platelet endothelial cell adhesion molecule-1. 11. We found abnormal angiogenesis in the yolk sacs and embryos as well as underdevelopment of branchial arches at around E9.5. Endothelial cells differentiated from embryonic stem (ES) cells lacking Foxo1 showed a morphologically abnormal response to exogenous vascular endothelial growth factor (VEGF), viz. a flat polygonal morphology in contrast to the elongated spindle-like shape of wild-type endothelial cells. These results suggest that FOXO1 plays a critical role in qualitatively controlling the VEGF signaling pathway and is necessary for normal development of the vascular system early in life. Targeted Disruption of the Foxo1 Gene—A 625-bp probe starting from the ATG codon of the mouse Foxo1 gene was used to screen a 129SVJ genomic library (Stratagene). Analysis of several overlapping clones revealed that the first coding exon including the ATG codon ends 625 bp after the ATG codon. Further phage analysis revealed that the rest of the coding exons are not located within at least 15 kb downstream from the first coding exon. To construct a targeting vector, a 5-kb EcoRV-NotI fragment whose 3′-end terminated 330 bp upstream of the ATG codon was used as the 5′-arm. A 4.2-kb loxP-PGKneo-poly(A)-loxP-FOXO1-3A-poly(A) cassette, in which FOXO1-3A is a constitutively active form of the FOXO1 protein in terms of transcriptional activity, was used to replace a 2.5-kb sequence including a 1-kb sequence 3′ to the first NotI site in the first exon and a 1.5-kb intronic sequence following the first exon. A downstream 1.1-kb PCR fragment was used as the 3′-arm. Finally, a 1.3-kb cassette containing the diphtheria toxin A chain gene was added to the 3′-end of the 3′-arm to facilitate negative selection. The plasmid was linearized with KpnI and electroporated into ES cells. Cells were selected with G418 (Invitrogen). G418-resistant clones were screened by PCR with primers for the sequence flanking the 1.2-kb short arm to confirm a corrective recombination of the 3′-arm. Normal (2.1 kb) and targeted (7.4 kb) loci were distinguished by NsiI digestion when probed with a 300-bp SacI genomic fragment for 3′-arm recombination (Fig. 1B). Normal (7.5 kb) and targeted (6.3 kb) loci were distinguished by XbaI digestion when probed with a 400-bp SacI-XbaI genomic fragment for 5′-arm recombination (Fig. 1B). Six correctly targeted ES cell clones were expanded, microinjected into C57BL/6J blastocysts, and transferred to the uteri of pseudopregnant ICR mice. Chimeric animals were backcrossed onto a C57BL/6J background to screen for germ line transmission. Chimeric males from two independent clones passed the mutant allele to offspring, and the animals from two lines showed identical phenotypes. Tail biopsy or yolk sack genomic DNA was amplified by PCR with primers specific for the wild-type allele and the target (wild-type Foxo1, 5′-GCAGAGATTAGCCTTCACGGCATG-3′ (upper) and 5′-TCTATGCTACAACTGATGGCATGG-3′ (lower); and targeted allele, 5′-TCTTCACAATGCCGCCTGAAAGGC-3′ (upper) and 5′-TCTATGCTACAACTGATGGCATGG-3′ (lower)). To confirm the deficiency of FOXO1 protein, Western blot analysis was performed for proteins of whole embryos at E9.5. They were lysed in radioimmune precipitation assay buffer (50 mm Tris-HCl (pH 7.4) containing 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, and 1 mm NaF), and aliquots of the proteins were separated on 7.5% reducing polyacrylamide gels and blotted onto Immobilon-P polyvinylidene difluoride membranes (Millipore Corp.). The blots were analyzed using a specific antibody against FKHR (Upstate Biotechnology, Inc.) and a standard chemiluminescence detection kit (ECL, Amersham Biosciences). The generation of ES cells homozygous for the targeted allele was performed using higher concentrations of G418 (18Mortensen R.M. Conner D.A. Chao S. Geisterfer-Lowrance A.A. Seidman J.G. Mol. Cell. Biol. 1992; 12: 2391-2395Crossref PubMed Scopus (354) Google Scholar). One cell line heterozygous for the targeted allele was cultured in medium supplemented with 1.2, 1.8, 2.4, 3.0, or 3.6 mg/ml G418. Thirty-three colonies were picked up from the plate with 3.6 mg/ml G418 after selection for 11 days and expanded. The genomic DNA was extracted and analyzed by Southern blotting using the probe specific for Foxo1 described above. Only one was cloned. In Vitro Differentiation of ES Cells—In vitro differentiation of ES cells was induced as described previously (19Matsumura K. Hirashima M. Ogawa M. Kubo H. Hisatsune H. Kondo N. Nishikawa S. Chiba T. Blood. 2003; 101: 1367-1374Crossref PubMed Scopus (49) Google Scholar). In brief, 5 × 104 ES cells were inoculated into a 25-cm2 culture flask preseeded with OP9 stromal cells and cultured for 5 days in induction medium (α-minimal essential medium supplemented with 10% fetal calf serum and 5 × 10-5 mol/liter 2-mercaptoethanol) in the absence of leukemia inhibitory factor. Cultured cells were dissociated with cell dissociation buffer (Invitrogen) and stained with fluorescein isothiocyanate-labeled anti-CD31 monoclonal antibody (mAb) (Pharmingen) and allophyco-cyanin-conjugated anti-vascular endothelial cadherin (VE-cadherin) mAb (20Matsuyoshi N. Toda K. Horiguchi Y. Tanaka T. Nakagawa S. Takeichi M. Imamura S. Proc. Assoc. Am. Physicians. 1997; 109: 362-371PubMed Google Scholar). CD31+ and VE-cadherin+ endothelial cells were sorted using a FACSVantage cell sorter (BD Biosciences). Then, 1 × 103 sorted endothelial cells/well were inoculated into either 6-well culture plates or 2-well culture slides preseeded with OP9 stromal cells. Cells were cultured for 3 days in induction medium in the presence or absence of exogenous human VEGF-A165 (Genzyme) and subjected to immunostaining of endothelial cell colonies. Reverse Transcription (RT)-PCR—Total RNA was purified from embryos and the yolk sacs of wild-type and Foxo1-null embryos at E9.5 (n = 3) using TRIzol (Invitrogen). After an RT reaction using Super-Script II (Invitrogen), quantitative PCR analysis of several genes involved in vasculogenesis and angiogenesis was performed using a QuantiTect SYBR Green PCR kit (QIAGEN Inc.); oligonucleotides specific for angiopoietin-1, angiopoietin-2, VEGF, Flt-1, Flk-1, Tie-1, Tie-2, EphB3, EphB4, ephrin-B2, connexin-37, connexin-40, connexin-43, and smooth muscle α-actin (Proligo Japan); and a Light Cycler system (Roche Applied Science). The following conditions were used: 95 °C for 15 min and 25 cycles at 94 °C for 10 s, 58 °C for 20 s, and 72 °C for 20 s. The amount of RT reaction product was controlled on the basis of the expression level of the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene. For confirmation of Foxo1 mRNA expression, RT-PCR for Foxo1 was performed using PCR primers specific for Foxo1 (upper, 5′-ACGAACTCGGAGGCTCCTTAGACAC-3′; and lower, 5′-GACTGGAGGTGGTCGAGTTGGACTG-3′) under the following conditions: 94 °C for 2 min; 35 cycles at 94 °C for 10 s, 63 °C for 30 s, and 72 °C for 45 s; and 72 °C for 2 min. RNA loading was controlled by amplification of the glyceraldehyde-3-phosphate dehydrogenase house-keeping gene. Negative controls were performed for each sample using non-reverse-transcribed RNA. Histology—For histological analysis, embryos were embedded in paraffin after fixation with 4% paraformaldehyde (PFA). Transverse sections were made at 7-μm intervals throughout the embryos and stained with hematoxylin/eosin solution. In Situ Hybridization—Whole mount in situ hybridization was carried out as described previously (21Yoshikawa Y. Fujimori T. McMahon A.P. Takada S. Dev. Biol. 1997; 183: 234-242Crossref PubMed Scopus (223) Google Scholar) with minor modifications. Embryos were collected in ice-cold phosphate-buffered saline (PBS), fixed in 4% PFA in PBS at 4 °C for 3 h, washed with PBS containing 0.1% Triton X-100 (PBST), dehydrated, and stored in methanol at -20 °C until used. After being bleached with 6% hydrogen peroxide in methanol for 2 h at room temperature, embryos were rehydrated though a 75, 50, 25, and 0% methanol and PBST series. Embryos were incubated with 20 μg/ml proteinase K in PBST for 6 min at E8.5∼9.0 or for 9 min at E9.5 and refixed in 4% PFA and 0.2% glutaraldehyde in PBS for 20 min. After incubation in hybridization buffer at 63 °C for >4 h, embryos were incubated in the same buffer containing 0.5 μg/ml digoxigenin-labeled RNA probe (Roche Applied Science) at 63 °C for 18 h. The hybridization buffer used and the steps for probe washing, RNase reaction, and RNase inactivation were as described previously (21Yoshikawa Y. Fujimori T. McMahon A.P. Takada S. Dev. Biol. 1997; 183: 234-242Crossref PubMed Scopus (223) Google Scholar). After incubation with 10% heat-inactivated goat serum and 2 mm levamisole (Sigma) in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST), embryos were treated with 0.24 units/ml anti-digoxigenin Fab fragments (Roche Applied Science) in TBST for2hat4 °C. After a thorough washing with 2 mm levamisole in TBST, embryos were treated with 0.1 m NaCl, 0.1 m Tris-HCl (pH 9.5), 20 mm MgCl2, 0.1% Tween 20, and 2 mm levamisole for 40 min, and hybridization products were visualized using BM Purple (Roche Applied Science) as a substrate. The Foxo1 and Crabp1 (cellular retinoic acid-binding protein-1) probe regions used in the in situ study were as described previously (14Furuyama T. Nakazawa T. Nakano I. Mori N. Biochem. J. 2000; 349: 629-634Crossref PubMed Scopus (541) Google Scholar, 22Maden M. Horton C. Graham A. Leonard L. Pizzey J. Siegenthaler G. Lumsden A. Eriksson U. Mech. Dev. 1992; 37: 13-23Crossref PubMed Scopus (102) Google Scholar). Immunochemistry—Whole mount immunohistochemistry was performed with mAb MEC13.3 (rat anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM-1) antibody, Pharmingen) as described previously (23Kataoka H. Takakura N. Nishikawa S. Tsuchida K. Kodama H. Kunisada T. Risau W. Kita T. Nishikawa S.-I. Dev. Growth Differ. 1997; 39: 729-740Crossref PubMed Scopus (179) Google Scholar, 24Nishikawa S.-I. Nishikawa S. Kawamoto H. Yoshida H. Kizumoto M. Kataoka H. Katsura Y. Immunity. 1998; 8: 761-769Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Embryos were fixed in 2% PFA and PBS at 4 °C for 1 h and dehydrated in methanol. Embryos were bleached in 5% hydrogen peroxide in methanol for 30 min at 4 °C. This was followed by rehydration and blocking in PBS containing 3% skim milk and 0.3% Triton X-100 (PBSMT) at 4 °C twice for 1 h. Embryos were incubated overnight in 10 μg/ml anti-mouse PECAM-1 antibody in PBSMT at 4 °C, washed with PBSMT at 4 °C, and incubated overnight with horse-radish peroxidase-conjugated antibody in PBSMT at 4 °C. Embryos were again washed with PBSMT at 4 °C and rinsed in PBST at room temperature for 20 min. For detecting signals, embryos were incubated in 0.3 mg/ml diaminobenzidine (Sigma) in PBST containing 0.5% NiCl for 20 min; hydrogen peroxide was added to a final concentration of 0.015%; and the embryos were incubated for 20–30 min. The staining reaction was terminated by rinsing the embryos in PBST. Embryos were post-fixed overnight in 4% PFA and PBS at 4 °C. To better observe the vasculature, embryos were dehydrated through graded solutions up to 100% methanol. For the immunochemistry of endothelial cell colonies, 6-well culture plates were fixed in 2% PFA and stained first with purified anti-VE-cadherin mAb (Zymed Laboratories Inc.)and then with horseradish peroxidase-labeled goat anti-rat IgG antibody (BIOSOURCE). VE-cadherin+ endothelial cell colonies were revealed by the diaminobenzidine/NiCl substrate. For confocal microscopy, 2-well culture slides were fixed in 2% PFA and stained with anti-VE-cadherin mAb. VE-cadherin were immunolocalized by Alexa Fluor 488-labeled goat anti-rat IgG (Molecular Probes, Inc.). Confocal microscopic images of endothelial cell colonies were taken using an Olympus Fluoview laser scanning microscope. Expression of Foxo1 in Embryos—Whole mount in situ hybridization at E8.5 revealed remarkable expression of Foxo1 in the neural crest cells migrating toward branchial arches and in the putative somites (Fig. 2A). The expression of Foxo1 was subsequently down-regulated and localized to the first and second branchial arches and putative somites at E9.0 and E9.5 (Fig. 2, B–E). The expression of Foxo1 was also observed in the intersomitic vessel, although it could not be determined whether the expression was localized to the endothelial cells (Fig. 2, D and E). The expression of Foxo1 in the yolk sac was below the detection limit of the in situ hybridization technique. However, RT-PCR confirmed the expression in the yolk sac at E9.5 (Fig. 2F). Targeted Inactivation of the Foxo1 Gene—The replacement vector disrupts the coding sequence by inserting a loxP-PGK-neo-poly(A)-loxP-FOXO1-3A-poly(A) cassette into the locus (Fig. 1, A and B). This deletes a portion of the first intron (including the splice donor) and the first 208 codons of the first coding exon, which encodes the N-terminal half of the forkhead domain of FOXO1. Mice heterozygous for the targeted allele were fertile and exhibited no obvious abnormalities. Heterozygous intercrosses yielded no viable offspring that were homozygous for the targeted allele (Foxo1-/-), consistent with a lethal embryonic phenotype (Table I). The first Foxo1-/- embryos were identified at E11.5, having been reabsorbed. Viable embryos were found at E10.5 with beating hearts, but with significant growth retardation. At E9.5, the distribution of Foxo1-/- embryos approached the expected mendelian frequencies (Table I). Analysis of embryonic mRNA by RT-PCR revealed Foxo1 transcripts of the expected length in wild-type embryos, but not in Foxo1-/- embryos (Fig. 1C). Because an ∼75-kDa band of FOXO1 protein detectable in wild-type embryos could not be identified in Foxo1-/- embryos by Western blot analyses (Fig. 1D), we concluded that no complete form of FOXO1 protein was synthesized in Foxo1-/- embryos.Table IGenotypes of mice resulting from matings of heterozygous FKHR+/-miceGenotypeFKHR+/+FKHR+/-FKHR-/-9.5 days postcoitusaGenotyping of three embryos in the 9.5-day postcoitus group was inconclusiveObserved293916Expected22442210.5 days postcoitusbGenotype of one embryo was inconclusiveObserved6169cEight of nine FKHR-/- embryos were abnormal and underwent absorptionExpected8168Live birthsObserved12170Expected7147a Genotyping of three embryos in the 9.5-day postcoitus group was inconclusiveb Genotype of one embryo was inconclusivec Eight of nine FKHR-/- embryos were abnormal and underwent absorption Open table in a new tab Abnormal Development of Branchial Arches and Aortic Arch Vessels in Foxo1-/-Embryos—At E9.0, wild-type and mutant embryos were indistinguishable in size; but after E9.5, Foxo1-/- embryos exhibited apparent growth retardation (Fig. 2, G and H). By E9.5, mutant embryos had developed a small first branchial arch, but no second branchial arch, and often exhibited marked pericardial swelling (Fig. 2, H and J). Since cranial neural crest cells significantly contribute to the formation of the branchial arches, we followed the pathway of crest cells using whole mount in situ hybridization with a Crabp1 probe as a specific marker for neural crest cells at E9.5. Specific staining in three well defined streams of representing cells migrating ventrally toward the arches could be seen in both wild-type and mutant embryos (Fig. 2, I and J). Histological analysis of transverse sections of E9.5 Foxo1-deficient embryos revealed that the dorsal aorta was severely underdeveloped and irregularly formed. Hypoplasia of branchial arches and aortic arch arteries was also observed (Fig. 2, K and L). To examine the extent of vascularization of Foxo1-deficient embryos, we performed whole mount immunohistochemical staining at E8.5–9.5 using a mAb against the endothelium-specific marker PECAM-1. We found that the differentiated endothelial cells were present at E8.5 at sites consistent with blood vessel development (Fig. 3A) and that the formation of the dorsal aorta was unaffected by the defect in the Foxo1 gene (Fig. 3B). However, there was no evidence of the aortic arch arteries arising from the aortic sacs in the mutant embryos (Fig. 3D), whereas the first and second aortic arch arteries were readily apparent in E9.5 wild-type embryos (Fig. 3C). The lack of aortic arch vessels most likely resulted in the severe cardiac failure as evidenced by the collection of a large pericardial effusion, with embryonic death occurring soon thereafter. Moreover, the capillary bed appeared dilated and apparently arrested at the primary plexus stage in the heads of mutant embryos (Fig. 3, E and F). Abnormal Vascular Remodeling in the Yolk Sacs of Foxo1-/-Embryos—The vitelline circulation in the embryonic yolk sac represents the earliest circulatory system and is the first site of vasculogenesis and angiogenesis in the embryo. As shown in Fig. 3 (G and H), the wild-type yolk sacs appeared to have a vasculature, but the Foxo1-deficient yolk sacs were pale and had no clear vasculature. The visceral endoderm and mesoderm forming the yolk sacs were not fused except at discrete foci, giving the yolk sacs of Foxo1 mutants a characteristic appearance (Fig. 3H), with large cavities present in the mutant yolk sacs that were lined by endothelial cells and that contained blood cells. PECAM-1 labeling revealed that endothelial cells were present in both wild-type and Foxo1-deficient yolk sacs at E8.75 (Fig. 3, I and J). Normally, a honeycomb-like vascular plexus was evident by E8.75 (Fig. 3I), and subsequent remodeling of the vasculature (angiogenesis) resulted in the formation of large vitelline vessels and a fine network of smaller vessels by E9.5 (Fig. 3K). Foxo1-deficient yolk sacs showed a honeycomb-like vascular plexus similar to that of wild-type yolk sacs at E8.75 (Fig. 3J), but failed to develop a normal vasculature at E9.5 (Fig. 3L). Histological analysis revealed that no distinct blood vessels were evident in the Foxo1-deficient yolk sacs (Fig. 3N), whereas endothelial cell-lined capillary-like vessels containing blood cells were seen in the wild-type yolk sacs (Fig. 3M). All together, the above evidence suggests that vasculogenesis (but not angiogenesis) proceeds without functional FOXO1. To determine whether other molecules contributed to the impaired angiogenesis in the mutants, we examined the expression of several factors and receptors involved in vasculogenesis and angiogenesis. By quantitative RT-PCR analysis, transcripts of VEGF, Flt-1, Flk-1, angiopoietin-1, angiopoietin-2, Tie-1, Tie-2, EphB2, EphB3, EphB4, connexin-43, connexin-45, and smooth muscle α-actin were detected at similar levels, with no statistical significance in wild-type and Foxo1-/- yolk sacs at E9.5 (Fig. 4). On the other hand, the expression levels of connexin-37, connexin-40, and ephrin-B2 in Foxo1-deficient yolk sacs were significantly reduced to ∼10, 1, and 35% of the wild-type levels, respectively (p < 0.01). It is possible that the vascular defects in the Foxo1-deficient yolk sacs are due in part to the reduction of expression of the specific connexins and ephrin among the molecules examined. However, we cannot eliminate the possibility of changes in unidentified gene expression in specific regions of mutant embryos. Expression of Foxo1 Transcripts in Endothelial Cells Derived from ES Cells—We demonstrated the presence of Foxo1 transcripts in the yolk sac (Fig. 2F), although the localization could not be determined by in situ hybridization. We examined the expression of the Foxo1 gene in the endothelial cell lineage using an in vitro differentiation system composed of ES cells. Foxo1+/+ and Foxo1-/- ES cells were co-cultured with OP9 stromal cells to induce differentiation of endothelial cells. Under the culture conditions used in this study, the frequency of endothelial cells among the differentiating ES cells was almost comparable between cultures initiated from Foxo1+/+ and Foxo1-/- ES cells (data not shown). CD31+ and VE-cadherin+ endothelial cells were purified using a fluorescence-activated cell sorter and subjected to RT-PCR analysis for the expression of several genes. Foxo1 transcripts were detected in the endothelial cells derived from Foxo1+/+ (but not Foxo1-/-) ES cells (Fig. 5A, panel a), suggesting that Foxo1 has some functional role in the endothelial cell lineage. Consistent with the observations in the yolk sac described above, transcripts of Flt-1, Flk-1, Flt-4, Tie-1, and Tie-2 were detected at similar levels in Foxo1+/+ and Foxo1-/- endothelial cells (Fig. 5A, panel b), indicating that the expression of these molecules does not depend upon Foxo1. Abnormal Behavior of Foxo1-/-Endothelial Cells in Response to VEGF—Endothelial cells derived from Foxo1+/+ and Foxo1-/- ES cells by co-cultivation with OP9 stromal cells were purified using a
Abstract This study demonstrates, for the first time, that loss of a single forkhead box class O (FoxO) transcription factor, can promote lymphomagenesis. Using two different mouse models, we show that FoxO3 has a significant tumour-suppressor function in the context of Myc-driven lymphomagenesis. Loss of FoxO3 significantly accelerated myeloid tumorigenesis in vavP- MYC10 transgenic mice and B lymphomagenesis in Eμ- myc transgenic mice. Tumour analysis indicated that the selective pressure for mutation of the p53 pathway during Eμ- myc lymphomagenesis was not altered. Frank tumours were preceded by elevated macrophage numbers in FoxO3 −/− vavP- MYC10 mice but, surprisingly, pre-B-cell numbers were relatively normal in healthy young FoxO3 −/− Eμ- myc mice. In vitro assays revealed enhanced survival capacity of Myc-driven cells lacking FoxO3, but no change in cell cycling was detected. The loss of FoxO3 may also be affecting other tumour-suppressive functions for which FoxO1/4 cannot fully compensate.
Human dental pulp stem cells (DPSCs) contain subsets of progenitor/stem cells with high angiogenic, neurogenic and regenerative potential useful for cell therapy. It is essential to develop a safe and efficacious method to isolate the clinical-grade DPSCs subsets from a small amount of pulp tissue without using conventional flow cytometry. Thus, a method for isolation of DPSCs subsets based on their migratory response to optimized concentration of 100 ng/ml of granulocyte-colony stimulating factor (G-CSF) was determined in this study. The DPSCs mobilized by G-CSF (MDPSCs) were enriched for CD105, C-X-C chemokine receptor type 4 (CXCR-4) and G-CSF receptor (G-CSFR) positive cells, demonstrating stem cell properties including high proliferation rate and stability. The absence of abnormalities/aberrations in karyotype and lack of tumor formation after transplantation in an immunodeficient mouse were demonstrated. The conditioned medium of MDPSCs exhibited anti-apoptotic activity, enhanced migration and immunomodulatory properties. Furthermore, transplantation of MDPSCs accelerated vasculogenesis in an ischemic hindlimb model and augmented regenerated pulp tissue in an ectopic tooth root model compared to that of colony-derived DPSCs, indicating higher regenerative potential of MDPSCs. In conclusion, this isolation method for DPSCs subsets is safe and efficacious, having utility for potential clinical applications to autologous cell transplantation.
