FoxO genes are dispensable during gastrulation but required for late embryogenesis in Xenopus laevis

Developmental Biology 337 (2010) 259–273

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Developmental Biology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / d e v e l o p m e n t a l b i o l o g y

FoxO genes are dispensable during gastrulation but required for late embryogenesis in Xenopus laevis Maximilian Schuff a, Doreen Siegel a, Nabila Bardine a, Franz Oswald b, Cornelia Donow a, Walter Knöchel a,⁎ a b

Institute of Biochemistry, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany Department of Internal Medicine I, University of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany

a r t i c l e

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Article history: Received for publication 20 May 2009 Revised 27 October 2009 Accepted 27 October 2009 Available online 3 November 2009 Keywords: Xenopus laevis FoxO genes Gastrulation Posttranslational modification Akt-1 Antisense morpholino oligonucleotide Heart Eye

a b s t r a c t Forkhead box (Fox) transcription factors of subclass O are involved in cell survival, proliferation, apoptosis, cell metabolism and prevention of oxidative stress. FoxO genes are highly conserved throughout evolution and their functions were analyzed in several vertebrate and invertebrate organisms. We here report on the identification of FoxO4 and FoxO6 genes in Xenopus laevis and analyze their expression patterns in comparison with the previously described FoxO1 and FoxO3 genes. We demonstrate significant differences in their temporal and spatial expression during embryogenesis and in their relative expression within adult tissues. Overexpression of FoxO1, FoxO4 or FoxO6 results in severe gastrulation defects, while overexpression of FoxO3 reveals this defect only in a constitutively active form containing mutations of Akt-1 target sites. Injections of FoxO antisense morpholino oligonucleotides (MO) did not influence gastrulation, but, later onwards, the embryos showed a delay of development, severe body axis reduction and, finally, a high rate of lethality. Injection of FoxO4MO leads to specific defects in eye formation, neural crest migration and heart development, the latter being accompanied by loss of myocardin expression. Our observations suggest that FoxO genes in X. laevis are dispensable until blastopore closure but are required for tissue differentiation and organogenesis. © 2009 Elsevier Inc. All rights reserved.

Introduction The mammalian FoxO subclass contains the members FoxO1 (FKH1, FKHR), FoxO3 (FKHRL1), FoxO4 (AFX, AFX1, MLLT7) and FoxO6. These genes represent homologues to the daf-16 gene in Caenorhabditis elegans, which is involved in nematode dauer formation. It was further shown that daf-16 plays a role in life span regulation as well as in insulin-like signaling (Lin et al., 1997). Since these functions were anticipated to be evolutionary conserved, FoxO genes have been intensively characterized in mammals. It has been shown that they play important roles in regulating genes involved in oxidative stress resistance, like the Sod-2 and the catalase gene (Honda and Honda 1999; Yanase et al., 2002; Tan et al., 2008). They also regulate genes involved in DNA damage repair (Gadd45a) (Tran et al., 2002), cell metabolism (glucose-6-phosphatase, phosphoenolpyruvate carboxykinase) (Puigserver et al., 2003; Hall et al., 2000), cell cycle arrest (p21CIP1, p27kip1) (Rathbone et al., 2008) and apoptosis (FasL, Bim) (Stahl et al., 2002; Lam et al., 2006). Fox factors contain a DNA binding winged helix domain and generally act as transcription regulators. The molecular mechanisms underlying the manifold functions mediated by FoxO proteins became even more complex by the finding that the activity and/or the

⁎ Corresponding author. Fax: +49 0 731/5023277. E-mail address: [email protected] (W. Knöchel). 0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2009.10.036

subcellular localization of FOXO proteins depend on various posttranslational modifications such as phosphorylation, acetylation and ubiquitination (Calnan and Brunet, 2008). Phosphorylation mediated by the highly conserved PI3K/Akt pathway is a well-characterized modification of FOXO proteins leading to their functional inhibition. There are three known sites within mammalian FOXO proteins being phosphorylated by Akt-1 and the related SGK (serum and glucocorticoid-induced kinase). Phosphorylation results in the binding to 143-3 chaperone proteins, sequestration in the cytosol and, finally, in degradation. Additionally, phosphorylation via CDK2 or IKKβ mediates inhibition of FOXO1 protein and negative transcriptional activity (Huang et al., 2006; Huang and Tindall, 2007). On the other hand, FoxO proteins have been shown to be phosphorylated by Jun Nterminal kinase (JNK) resulting in accumulation of FOX transcription factors within the nucleus. Furthermore, cytosolic retention can be inhibited by phosphorylation via MST1 (Lehtinen et al., 2006; Huang and Tindall, 2007). Acetylation and deacetylation of FOXO factors represent another regulatory mechanism for their activity. The SIRT1 genes, also known as sirtuins or HDAC class III, are a widespread family of NAD+dependent protein deacetylases. They are involved in the cellular response to stress and in changes of the status of nutrition. The SIRT1 proteins form complexes with FOXO transcription factors (Brunet et al., 2004). Many studies demonstrated that elevated SIRT1 deacetylase activity increases longevity in nematodes and flies (Guarente, 2007). Interestingly, the interaction of sirtuins and FOXO factors

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inhibits NF-κB signaling, thereby protecting against inflammatory and aging processes, and enhances the expression of target genes, which are involved in DNA repair and cell cycle arrest (Salminen et al., 2008).

In addition, monoubiquitination of FOXO4 in response to oxidative stress increases the nuclear protein level and transcriptional activity, but the detailed mechanism of this monoubiquitination process is still

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not clear. However, deubiquitination is probably performed by the deubiquitinase USP7/HAUSP (van der Horst et al., 2006). Loss of FOXO function has also been identified in several human cancers. FOXO genes are involved in tumor associated chromosomal rearrangements. Two fusions of FOXO1 with Pax3 or Pax7 were found being linked with alveolar rhabdomyosarcomas (Galili et al., 1993), while fusions of FOXO3 or FOXO4 with the MLL gene were found in secondary acute leukemia (So and Cleary, 2003). Functional differences of mammalian FoxO genes became apparent by knockout experiments. Homozygous Foxo1 knockout mice die at stage E10.5 due to vessel defects and abnormal angiogenesis (Hosaka et al., 2004; Furuyama et al., 2004). The embryos reveal reduced size and retarded cardiac looping. Transgenic mice expressing a constitutive active form of Foxo1 in the liver and pancreatic β-cells reveal a failure in β-cell development, glucose intolerance and diabetes in an age dependent manner, indicating an important role of Foxo1 in the insulin pathway (Nakae et al., 2002). Foxo3 knockout mice do not exhibit obvious physiological abnormalities. However, Foxo3(−/−) females are infertile and reveal abnormal follicle development (Castrillon et al., 2003; Hosaka et al., 2004). Moreover, histological examination revealed widespread organ inflammation and hyperactivated helper T cells, indicating that a lack of Foxo3 signaling increases NF-κB activity (Lin et al., 2004). Foxo4 deficient mice do also not exhibit any significant phenotype. The Foxo6 gene has been intensively characterized (Jacobs et al., 2003; van der Heide et al., 2005), but no loss of function data are available so far. To further explore the role of FoxO genes in vertebrate embryonic development, we made use of Xenopus as a suitable model system to perform gain and loss of function studies. We have recently identified FoxO1 and FoxO3 in Xenopus laevis, and described the temporal and spatial expression patterns (Pohl et al., 2004). In the present report we characterize FoxO4 and FoxO6. By comparative overexpression and morpholino based knockdown studies of all Xenopus FoxO genes, we demonstrate that FoxO depletion does not influence gastrulation and blastopore closure but interferes with late development. High FoxO activity prevents gastrulation and directs cells towards a primitive ectodermal fate. Moreover, we here demonstrate that FoxO4 is required for proper heart, eye and craniofacial development. Materials and methods

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TACTTGTA-3′ (reverse complementary of translation start is underlined). For control, an antisense morpholino oligonucleotide derived from the human β-globin gene (Gene Tools, CoMO: 5′CCTCTTACCTCAGTTACAATTTATA-3′) was used. Additionally, a mismatch morpholino oligonucleotide for FoxO4 containing seven mismatches (mm-FoxO4MO: 5′-GCACTCGTTCACTTCGTTCCTTCGC-3′) was used as negative control. To test the specificity of the morpholinos in vitro, 0.5 μg of plasmid DNA was transcribed/translated using the TNT Coupled Reticulocyte Lysate Systems (Promega) and 35S labeled methionine in the absence or presence of 10 ng MO. Furthermore, translation of FoxO RNA was monitored in vivo using green fluorescent protein (GFP) fusion constructs. Either the 5′-region of the FoxO RNA (FoxO1, FoxO4 and FoxO6) or the full coding region (FoxO3) including the complete morpholino binding site were fused to GFP (Schuff et al., 2007). RNA was injected alone, together with FoxOMO or CoMO at different doses. Embryos were analyzed by fluorescence microscopy at stage 26. RT-PCR Total RNA was extracted from X. laevis embryos at indicated developmental stages using QIAzol (Qiagen), submitted to DNase I treatment and cleanup with RNeasy kit (Qiagen). First strand cDNA was synthesized from 2 μg of total RNA using RevertAid™ First Strand cDNA Synthesis kit (Fermentas). Primers are listed in supplementary Table S1. The real time RT-PCR was performed as previously described (Cao et al., 2008). Data are presented as relative units. DNA transfection and imaging HeLa cells (ATCC CCL2) were grown at 37 °C under 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS). For imaging, cells were cultured on chambered coverslips (Nunc) at a density of 105 cells per cm2. After 16 h, cells were transfected with 500 ng of expression plasmids using the Nanofectin transfection reagent (PAA). The living cells were analyzed 24 h after transfection using a fluorescence microscope (IX71, Olympus) equipped with a digital camera (C4742, Hamamatsu), a 100-W mercury lamp (HBO 103W/2, Osram) and the following filter set: ex: HQ470/40, em: HQ525/50. Leptomycin B (Sigma, 2.5 ng/ml) was added to cells 50 min prior to imaging.

Database analyses and alignments Cloning of genes and in vitro transcription Sequence analyses, EST and genomic database search and multiple sequence alignments were done using the following bioinformatic programs: http://www.ncbi.nlm.nih.gov/blast; http://www. ensembl.org/index.html; http://www.ebi.ac.uk/clustalw/. Antisense morpholino oligonucleotides Antisense morpholino oligonucleotides (MO) directed against the translation start sites of FoxO1, FoxO3, FoxO4 and FoxO6 were analyzed by the mfold database to minimize secondary folding structures (http:// www.bioinfo.rpi.edu/applications/mfold/) and purchased from Gene Tools. FoxO1MO: 5′-AGGAGCCTCAGCCATGGTTTCAACT-3′. FoxO3MO: 5′-TCTGCCATGCTGCGAGAAGGGTCTT-3′. FoxO4MO: 5′-GGACAGGTTCAGTTCCTTCCATGGC-3′. FoxO6MO: 5′-GCTTCCAGCTTCTCCAT-

Genes were amplified from cDNA or cDNA libraries with Taq Polymerase (ABgene) and cloned in pDrive vector (Qiagen) or amplified with Reddy-Mix (Abigene) and oligonucleotides including the restriction sites. The mutations in FoxO3-TM and Akt-DM (T309E/ S474E) plasmids were introduced via PCR using mutated primers. Cloning primers used for the insertion of FoxO into the pCS2 vector were: Xenopus tropicalis FoxO1: F 5′-ATGGCTGAAGCGCCTCTGCCCC-3′, R 5′-AGGGTCTTTGTGAACACCATTTA-3′; X. tropicalis FoxO3: F 5′ATGGCAGAAGCCGTGCCTTCCCT-3′, R 5′-CATGTTATCTTCCCAGGCTTTATTC-3′; X. laevis FoxO4: F 5′-ATGGAAGGAACTGAACCTGTCCCC-3′, R 5′-GGCCAGAAATAAATCTCAAGGTTTTC-3′; X. laevis FoxO6: F 5′ATGGAGAAGCTGGAAGCTGAATCA-3′, R 5′-GCCCTACACATATATACTGGTTCAA-3′.

Fig. 1. FoxO genes in Xenopus. (A) Alignment of X. laevis and X. tropicalis FoxO4 and FoxO6 amino acid sequences. Identical amino acids (three out of four) are shown in red, the forkhead domain is shown in yellow. Putative phosphorylation sites are numbered in brackets and lysines, which are known to be acetylated in their mammalian homologues, are shaded in blue. (B) Schema of putative phosphorylation sites in FoxO proteins of X. laevis. Kinases and target sites are shown according to Calnan and Brunet (2008). Red boxes denote serine (S) or threonine (T) residues that are found to be phosphorylated within mammalian orthologous. Orange boxes show amino acids only known to be phosphorylated in paralogues, but conserved positions suggest their potential use for phosphorylation. (C) Genomic organization of FoxO genes in X. tropicalis. Mapping of gene structures was analyzed by the Ensembl database (X. tropicalis genome assembly version 4.1). Exons are numbered with Roman numerals. The coding sequence is shaded in grey, UTR's are shown in black. (D) Phylogenetic tree of FoxO proteins of different species created by ClustalW (c.e., Caenorhabditis elegans; h.s., Homo sapiens; m.m., Mus musculus; x.l., Xenopus laevis ). Corresponding accession numbers are given.

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For rescue experiments of FoxO4 depletion, we introduced 7 silent mutations in the MO binding site via PCR using mutated primers. The ORF was fused to the human glucocorticoid receptor (GR) ligand-binding domain (Gammill and Sive, 1997), referred to as FoxO4-GR. For activation of the GR fusion protein, dexamethasone (DMS; Sigma) was prepared as 5 mM stock solution in 100% ethanol and applied to control and RNA injected embryos at stage 8.5 in a concentration of 10 μM in 0.1 × MBSH. Embryos were kept in DMS until fixation. For overexpression studies, all injection constructs were linearized with NotI and transcribed with mMessage mMachine SP6 kit (Ambion). Embryo manipulation and in situ hybridization In vitro fertilization and embryo culture were done according to standard protocols. Embryonic stages were determined according to Nieuwkoop and Faber (1967). Whole mount in situ hybridizations were performed according to standard procedures (Harland, 1991). To determine the injected side after unilateral injections, we either coinjected fluorescein dextrane or lacZ RNA (200 pg). LacZ staining was performed according to standard protocols. Alcian blue staining and sectioning Alcian blue staining was performed as previously described (Schuff et al. 2007). For sectioning, embryos were embedded in agarose, in gelatine/albumine or paraffin according to standard protocols. Serial vibratome or paraffin sections (10–50 μm) were processed. BrdU, TUNEL and caspase assay BrdU and TUNEL assays were performed as previously described (Schuff et al., 2007). For gain of function 10 nl BrdU labeling reagent (Roche) was injected at stage 9 and 11 in the animal region. Embryos were fixed at stage 9.5 or stage 11.5, respectively. To determine caspase activities, embryos were collected at stage 11.5, stage 19 or stage 26 and subjected to caspase 3/7, caspase 8 and caspase 9 Glo Assay kits (Promega) following the manufacturer's protocol and modifications as described (Boorse et al., 2006). Each 10 embryos were homogenized in 200 μl phosphate buffered saline (PBS). Results Isolation and cloning of FoxO4 and FoxO6 Xenopus FoxO4 was first identified in two ESTs of X. tropicalis (accession no. DR845397 and ES676911) and in two ESTs of X. laevis (accession no. CK805952 and DC117457). By PCR we amplified a 1746 bp fragment from X. laevis stage 29 cDNA, which encodes the complete open reading frame, since the start codon is preceded by a stop codon. X. laevis FoxO4 cDNA sequence was deposited under EMBL accession no. FJ811896. The corresponding protein contains 485 amino acids sharing 52% identity with human and 51% identity with mouse FoxO4 protein, respectively. The X. tropicalis FoxO4 sequence was deduced from the genomic sequence (Ensembl database, version 4.1). Also, the FoxO6 gene of X. tropicalis containing the complete open reading frame and two ESTs (accession no. CR586109 and CR586113) are found in the database. The entire coding sequence of X. laevis was amplified from cDNA mixed from different developmental stages of X. laevis embryos using primers derived from the X. tropicalis 5′- and 3′-UTRs. X. laevis FoxO6 cDNA contains 1732 bp and was deposited under EMBL accession number FJ811897. The protein comprises 562 amino acids and exhibits 36% identity to the human and 39% to the mouse protein, respectively. Fig. 1A shows an alignment of X. laevis and X. tropicalis FoxO4 and FoxO6 proteins.

The identity between the orthologues accounts for 95%, between the paralogues it only amounts for 40% and is mainly restricted to the forkhead domain. Fig. 1B presents a schema of known or predicted phosphorylation sites within FoxO proteins including the serine/ threonine kinases specifically interacting with these target sites. Surprisingly, most amino acids that are phosphorylated in mammals are not only conserved in Xenopus orthologues but also within paralogues. We then have analyzed the organization of FoxO genes in the genome of X. tropicalis (Ensembl, version 4.1). FoxO1 is found in scaffold_233, FoxO3 in scaffold_269, the homologue of the human Xchromosomal located FOXO4 gene is located in scaffold_111 and FoxO6 in scaffold_478 (Fig. 1C). The four FoxO genes of X. tropicalis are composed of only two or three exons. Interestingly, x.t.FoxO1 as well as x.t.FoxO3 contain a separate exon in the 3′-untranslated region. We checked the genomic organization of human and mouse FOXO genes in the Ensembl database and found a 3′-untranslated exon as well (data not shown). As we cannot find any additional FoxO homologues in the Xenopus genome, we assume that Xenopus has the same number of FoxO genes like human and mouse, contrary to zebrafish or the southern platyfish having an additional foxo5 gene. The restricted existence of foxo5 to fish suggests that the underlying gene duplication event has occurred after divergence of reptiles and fishes. Fig. 1D shows a phylogenetic tree of known FoxO proteins from indicated species. Vertebrate FoxO proteins can be classified into two groups, one containing FoxO1 and FoxO4, the other containing FoxO3 and FoxO6. The FoxO proteins of X. laevis are rather close to their homologues from other species including the daf-16 protein of C. elegans, which is the only FoxO homologue known in this species. The identity between daf-16 and all FoxO proteins of X. laevis is almost 30% (data not shown), a rather high value regarding the evolutionary distance. FoxO genes show different expression patterns during embryogenesis and in adult tissues The temporal expression patterns of FoxO genes during Xenopus embryogenesis and their relative expression in different adult tissues were analyzed by real time RT-PCR (Fig. 2A). While FoxO1 is only weakly expressed until stage 12 and is highly upregulated in postgastrula embryos, FoxO3 shows a strong maternal expression. Transcripts decrease until the end of neurulation but are gradually increased from stage 20 onwards. FoxO4 can be detected maternally, but is also downregulated until gastrulation. At stage 12, FoxO4 is upregulated showing a second burst of expression at stage 45. FoxO6 shows a similar temporal pattern like FoxO1, and is upregulated from stage 20 onwards. A quite heterogenous distribution of FoxO transcripts was observed in different adult tissues. FoxO1 and FoxO3 are mainly expressed in gut and liver. FoxO4 transcripts are enriched in heart, brain and eye, whereas FoxO6 expression is mainly restricted to brain and eye. This finding correlates to previous results obtained from mice, where Foxo6 was shown to be predominantly expressed in the brain (Jacobs et al., 2003). The spatial expression patterns of FoxO4 and FoxO6 were analyzed by whole mount in situ hybridization. Expression of FoxO4 can hardly be detected at early cleavage stages (Fig. 2B). During neurula stages, staining is visible in the neural plate (Fig. 2C) and, later onwards, in the migrating neural crest cells as well as in the early eye field (Fig. 2D). Moreover, there is weak staining expanding the whole embryo (Figs. 2C and D), whereas no staining is observed in a control using a sense probe for in situ hybridization (Fig. 2E). At stage 29, FoxO4 transcripts are detected in the head, predominantly in the otic vesicle, the branchial arches and the presumptive retinal layer (Fig. 2F). At stage 35, FoxO4 shows also diffuse expression in the head and staining in the region of pronephros and pronephric duct (Fig. 2G). A transversal section of an embryo at stage 38 reveals FoxO4 transcripts

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within the eye, head mesenchyme and neural tube (Fig. 2G). Retinal expression is restricted laterally to the lens, where retinal cells are still not differentiated (Fig. 2G). Double in situ hybridization using the lens

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marker crystallin reveals that FoxO4 expression is absent from the lens (data not shown). FoxO6 expression is first detected in the hatching gland at stage 27 (Fig. 2I–K), as it also was reported for FoxO3 (Pohl et al., 2004). From stage 29 onwards, the distribution of FoxO6 transcripts is quite diffuse in the branchial arches and in the head (Fig. 2L), with two expression domains of higher intensity being visible in the olfactory pits (Fig. 2M). This observation might be correlated with previous findings in mice, where Foxo6 transcripts were also detected in the olfactory epithelium of E14.5 embryos and in the medial part of the anterior olfactory nucleus of adults (Jacobs et al., 2003). Overexpression of FoxO genes causes severe gastrulation defects To investigate the role of Xenopus FoxO genes during development we first performed gain of function studies by injection of X. laevis FoxO1, FoxO3, FoxO4, FoxO6 or C. elegans daf-16 RNA at different amounts into 2-cell stage embryos. In case of FoxO1, FoxO4, FoxO6 and daf-16, but not in case of FoxO3, we observe severe gastrulation defects in a dose dependent manner. While FoxO1 or daf-16 injected embryos survived until stage 24 even at high RNA dose (500 pg/ blastomere), bilaterally FoxO6 RNA injected embryos (200 pg/ blastomere) do not even form a blastopore. Most of these embryos die around stage 14. In case of FoxO4 RNA injected embryos, only fragments of the blastopore are formed, coupled with a high rate of mortality around stage 11. Fig. 3A (right column) shows the phenotype of unilaterally injected embryos at stage 18, resulting in open neural folds. Interestingly, embryos injected with FoxO3 RNA develop normally (see Fig. 4B). To investigate the molecular basis of the gastrulation phenotype, we have analyzed FoxO RNA injected embryos by whole mount in situ hybridization using different markers (Fig. 3A). Except for FoxO1, we observe a downregulation of the panmesodermal marker Xbra and of the endodermal marker Sox17α at stage 11. Performing lacZ staining we found a correlation between the sites of lacZ expression and of lacking Xbra expression (data not shown). We also detect a severe decrease of the neural marker Chordin, when analyzed at stage 15. Analysis of the ventral marker XVent2B or the dorsal marker goosecoid (gsc) revealed also reduced expression (data not shown). Sox2, which both detects pluripotent cells until gastrula stage and later represents a neuroectodermal marker, is strongly reduced and almost absent in FoxO6 injected embryos (Fig. 3A). Interestingly, Xema (Xenopus ectodermallyexpressed mesendoderm antagonist (Suri et al., 2005), FoxI1e) is highly upregulated in FoxO4 and FoxO6 and slightly upregulated in FoxO1 and daf-16 injected embryos. To verify these observations, we also analyzed FoxO injected embryos by real time RT-PCR. The results support the data obtained by in situ hybridization. Additional markers tested by real time RT-PCR reveal a general downregulation of mesodermal (BMP4, XVent1, gsc, Siamois, Xnr6) and endodermal (Mixer) marker gene expression. We also observed inhibition of early neuroectodermal (XSox2, XSox3 and geminin H) and of late neural markers (N-tubulin and Ncam). Besides Xema we find an upregulation of Grhl1 that is restricted to cells with epidermal fate (Tao et al., 2005). Fig. 2. Expression of FoxO genes. (A) Temporal expression patterns of FoxO1, FoxO3, FoxO4 and FoxO6 genes during Xenopus embryogenesis and relative expression in adult tissues. Amount of transcripts was analyzed by semi-quantitative RT-PCR using RNA isolated from embryos at indicated developmental stages (Nieuwkoop and Faber, 1967) or from adult tissues. Expression levels are shown in relative units. Histone H4 was used as internal control. (B-M) Spatial patterns analyzed by whole mount in situ hybridization of FoxO4 (BH) and FoxO6 (I-M). (B) 2-cell stage, lateral view. (C) Stage 17, anterior view. (D) Stage 21, anterior view. (E) Stage 23 embryo hybridized with a FoxO4 sense probe, anterior view. (F) Magnification of the head of an embryo, stage 29. (G) Stage 35/36, lateral view. (H) Transversal section of the head, stage 38. (I) Stage 22, anterior view. (J) Stage 27, lateral view. (K) Magnification of the head at stage 28, anterior view. (L) Stage 32, lateral view. (M) Stage 30, anterior view. Red arrows indicate the eye field, yellow arrows indicate the olfactory pits. ba, branchial arches; hg, hatching gland; hm, head mesenchyme; l, lens; nt, neural tube; ov, otic vesicle; pd, pronephric duct; pl, presumptive lens; pr, presumptive retina; r, retina.

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Fig. 3. Overexpression of FoxO1, FoxO4, FoxO6 and daf-16 interferes with early Xenopus development. (A) Whole mount in situ hybridization of uninjected control embryos and FoxO RNA injected embryos for Xbra, Sox17α, Sox2 at stage 11 (vegetal view), Xema, stage 11 (animal view), and Chordin at stage 15 (dorsal view). RNAs were bilaterally injected at 2-cell stage with 500 pg/blastomere (FoxO1), 400 pg/blastomere (FoxO4), 200 pg/blastomere (FoxO6) and 400 pg/blastomere (daf-16). The right column shows the phenotypes of unilaterally FoxO RNA injected embryos at stage 18, dorsal view. (B) Real time RT-PCR in gain of function embryos. Injected embryos were collected at stage 11.5. RT-PCR was performed for indicated genes. Percentages of phenotypes are given in supplementary Table S2.

However, since FoxO4 and FoxO6 RNA injections prevent upregulation of E-cadherin and epidermal keratin, differentiation of epidermis seems also to be blocked. Noteworthy, we observe an upregulation of Oct60, a functional homologue to the pluripotency associated Oct3/ 4 factor in mammals (Cao et al., 2006).

FoxO3 becomes functionally activated by removal of Akt phosphorylation sites As overexpression of FoxO3 does not yield a phenotype (Fig. 4B), we further investigated whether this factor might be otherwise

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Fig. 4. FoxO overexpression phenotype depends upon Akt phosphorylation status. (A) Uninjected control embryo at stage 13. (B) Bilateral injection of 500 pg FoxO3 RNA does not reveal major phenotypic alterations. (C, D) Bilateral injection of 250 pg (C) or 500 pg FoxO3-TM RNA (D) prevents blastopore closure. (E) Uninjected control embryo at stage 11.5. (F) Bilateral injection of 400 pg FoxO4 prevents blastopore formation. (G) Bilateral injection of 300 pg IGF-1. (H) Co-injection 300 pg IGF-1 and 400 pg FoxO4 RNA restores blastopore formation. (I) Bilateral injection of 400 pg IGF-2. (J) Embryo, co-injected with 400 pg FoxO4 and 400 pg IGF-2 RNA, is rescued to wild type. (K) Bilateral injection of 500 pg Akt-1 RNA. (L) Embryo, co-injected with 400 pg FoxO4 and 500 pg Akt-1 RNA, is rescued to wild type. (M) Embryo, co-injected with 400 pg FoxO3-TM and 500 pg Akt-1 RNA, stage 11.5. The wild type phenotype cannot be restored. (N) Bilateral injection of 200 pg FoxO6 prevents blastopore formation. (O) Embryo, co-injected with 200 pg FoxO6 and 500 pg Akt-1. Blastopore formation fails. (P) Uninjected control embryo, stage 19. (Q) Bilateral injection of 500 pg Akt-1 RNA. (R) Embryo, co-injected with 400 pg FoxO4 and 500 pg Akt-1 RNA. Akt-1 prevents formation of the FoxO4 phenotype and allows neural plate formation. All bilateral injections were performed at 2-cell stage. (S) HeLa cells transfected with GFP, FoxO3-GFP, FoxO3-TM-GFP and FoxO6-GFP fusion-constructs. Percentages of blastopore closure are given in supplementary Table S3.

regulated regarding its exclusion from the nucleus and inactivation as transcriptional regulator. To examine the functional significance of the FoxO3 phosphorylation status, we mutated the three putative Akt1 phosphorylation sites (T30, S238 and S300) to alanine (ArimotoIshida et al., 2004). This triple mutated protein should not be shuttled to the cytosol but remains constitutively active in the nucleus. In fact, the injection of triple mutated FoxO3 (FoxO3-TM) RNA leads to similar gastrulation defects as FoxO1, FoxO4, FoxO6 or daf-16 (compare Figs. 4C, D with Fig. 3A). We next have asked whether phosphorylation by

Akt-1 kinase might generally exclude FoxO proteins from the nucleus suggesting that a higher level of Akt-1 can restore normal blastopore formation in FoxO overexpression. The Akt-1 gene of X. laevis has already been described (Andersen et al., 2003) and we have cloned the Akt-1 cDNA from X. tropicalis (accession no. BC123001). Since the IGF-1 receptor activates the phosphoinositol-dependent kinase-1/Akt signal transduction pathway (Foulstone et al., 2005), we have also coinjected FoxO4 with IGF-1 and IGF-2 RNA. Injection of 300 pg IGF-1 or 400 pg IGF-2 RNA does not influence gastrulation, while injection of

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400 pg FoxO4 RNA results in lack of a blastopore formation (Figs. 4F, G and I). Co-injection of IGF-1 or IGF-2 with FoxO4 RNA restores formation and closure of the blastopore (Figs. 4H and J). Injection of 1 ng Akt-1 RNA alone does not yield any effect either at stage 11.5 or at stage 19 (Figs. 4E, K, P, Q). 500 pg Akt-1 RNA are sufficient to restore blastopore formation and neurulation, while all embryos injected with 400 pg FoxO4 RNA alone or co-injected with a lower dose of Akt1 RNA (125 pg or 250 pg, data not shown) failed in gastrulation and died between stage 11 and 13 (Figs. 4F, L, R). However, co-injection of Akt-1 with FoxO3-TM or FoxO6 RNA cannot restore the wild type phenotype (Figs. 4M, O) and injected embryos die until the onset of neurulation. To explore whether missing Akt-1 phosphorylation sites prevent cytosolic shuttling, we performed an analysis of subcellular localization by transfection of HeLa cells with green fluorescent protein (GFP) fused to FoxO3, FoxO3-TM and FoxO6 (Fig. 4S). While FoxO3 is located in the cytoplasm, FoxO3-TM and FoxO6 are located within the nuclei. However, when nuclear export is blocked by incubation of cells with Leptomycin B, FoxO3 is also accumulated within the nucleus. This indicates that the missing overexpression phenotype for FoxO3–in contrast to other FoxO proteins–is mainly due to its cytoplasmic localization. Functional knockdown of FoxO genes leads to malformed embryos To analyze FoxO function in detail, we have performed loss of function experiments by injection of morpholino antisense oligonucleotides against all four FoxO genes (O1MO–O6MO). First we tested morpholino function in vitro by using a coupled transcription/translation kit and 35S methionine for labeling of proteins. Addition of morpholinos efficiently blocks translation of FoxO-GFP fusion-constructs (Fig. S1). As negative controls we used rescue constructs (see below) that should not be blocked by addition of MOs. For FoxO1 and FoxO3 we used the X. tropicalis RNA containing either mutations or only the coding sequence but not the 5′-UTR (Fig. S1A). For FoxO4 we introduced six silent mutations and for FoxO6 we mutated only the morpholino binding site into the corresponding sequence of X. tropicalis. In all these cases, addition of morpholinos (O1MO–O6MO) does not inhibit in vitro translation. For the in vivo test we used morpholino specific GFP sensors. RNA of these constructs was bilaterally injected into 2-cell stage embryos and fluorescence of embryos was documented at stage 26 (Fig. S1B). Co-injection of control morpholino (CoMO) does not decrease fluorescence, whereas injection of corresponding FoxOMO completely extinguishes green fluorescence. Additionally, co-injection of heterologous FoxOMO and FoxO-GFP RNA (for example co-injection of FoxO3MO with FoxO1-GFP RNA) has no influence on fluorescence (data not shown). These results indicate that the FoxOMOs are specific and inhibit efficiently the translation of corresponding RNAs. Next we have analyzed the phenotypes of embryos obtained after injection of FoxO1, FoxO3, FoxO4 and FoxO6 morpholinos. In all cases we observe normal closure of blastopore and, except for FoxO3, embryos exhibit almost normal development until the end of neurulation. Defects became apparent at later stages of development. Injection of FoxO1MO into both blastomeres of 2-cell stage embryos leads to developmental retardation and body deformation at stage 39 (Fig. 5A). When compared to CoMO injected embryos, FoxO1 depleted embryos are reduced in size, an observation that correlates to the reported FoxO1 knockout in mice (Hosaka et al., 2004). This size reduction, which is also visible after depletion of FoxO4 and, especially, of FoxO6, results in a reduced body axis and a crippled phenotype (Fig. 5A). The embryos do not survive and die between stages 43 and 46. Additionally, a mismatch morpholino for FoxO4 containing 7 mismatches (mm-FoxO4MO) was used as control. As expected, injection of 40 ng per blastomere at 2-cell stage has no influence on embryogenesis (Fig. 5A).

The broad phenotype observed after FoxO depletions suggests some general inhibitory mechanism. It is known that FoxO proteins regulate genes that are indispensable for life, such as the Sod-2 gene (Honda and Honda 1999; Yanase et al., 2002). In FoxO1, FoxO4 and FoxO6 depleted embryos, we found by RT-PCR at stage 18 and stage 24 a severe reduction of Sod-2 expression (Fig. 5B). Since the knockout of murine Sod-2 results in lethality (Li et al., 1995; Lebovitz et al., 1996), observed downregulation might contribute to the broad deficiencies of FoxO depletion phenotypes. Interestingly, injection of FoxO3MO into both cells of 2-cell stage embryos leads to blastopore closure but results in an early death during neurulation, whereas injection of CoMO has no effect on stage 19 embryos (see Fig. 5C). To check the specificity of the morpholinos, we have also rescued the loss of function phenotype by co-injection of FoxO RNA from X. tropicalis. 500 pg FoxO3 RNA were sufficient to render neural plate formation and to prevent death of embryos (Fig. 5C). We have also rescued the loss of function phenotype of FoxO4. As co-injections of FoxO4 RNA and FoxO4MO result either in gastrulation defects (GOF phenotype) or, at lower RNA concentrations, in the FoxO4 depletion phenotype, we tried to overcome this problem by using an inducible FoxO4-GR fusion construct, which contains seven silent mutations in the MO binding site, and analyzing eye rescue in injected embryos. While injection of 15 ng FoxO4MO in one dorsal blastomere at 4-cell stage inhibits eye development, injection of 15 ng CoMO does not influence eye formation. However, co-injection of 160 pg FoxO4-GR RNA restored the eye, when embryos were incubated in 10 μM dexamethasone (Fig. 5D). FoxO4MO injection affects formation of the eye, heart and craniofacial structures The phenotype of FoxO4 depleted embryos appeared to be more specific by showing distinct malformations of eye, heart and neural crest derivatives in a dose dependent manner. By bilateral injection of 20 ng FoxO4MO the eye is reduced but still visible (Fig. 5A), whereas unilateral injection of 40 ng FoxO4MO leads to an almost complete absence of eye structure on the injected side at stage 39 (Fig. 6A). This result correlates to the observed FoxO4 expression in the early eyefield (Figs. 2D and F). To analyze genetic dysregulations underlying this phenotype we performed in situ hybridizations with unilaterally FoxO4MO injected embryos for marker genes. Fig. 6A shows control and unilaterally FoxO4MO injected embryos at stage 21 stained for the early eye marker Pax-6. The expression domain at the injected side is strongly reduced. We observed a similar reduction of the early eye and forebrain marker Otx-2 in FoxO4 depleted embryos at stage 25 (Fig. 6A). Pax-6 expression could be restored by co-injection of FoxO4-GR RNA (see Fig. S2A) confirming the result of Fig. 5D. Since FoxO4 transcripts are also visible in the migrating cranial crest cells and the branchial arches (Fig. 2), we further investigated the expression of neural crest markers FoxD3 and AP2α in unilaterally FoxO4MO injected embryos (Fig. 6B). In contrast to the uninjected side, we detected loss or reduction of FoxD3 and AP2α expression at the most ventral population of migrating crest cells indicating a failure of cranial crest cell migration. As these cells do normally contribute to craniofacial cartilage formation, we expected corresponding malformations. Indeed, Alcian blue staining of control and unilaterally FoxO4MO injected embryos at stage 47 revealed severe reductions of the craniofacial cartilage (Fig. 6B). Especially the branchial arches are almost absent at the injected side. Table S2C represents percentages of lacking eye phenotypes and cartilage deformations in correlation to the amount of unilaterally injected FoxO4MO. Another distinct phenotypic feature became visible in the heart region of bilaterally FoxO4MO injected embryos at around stage 40, being regularly accompanied by oedemas (Fig. 6C). Percentages of heart phenotype in

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Fig. 5. Loss of FoxO function results in severe developmental defects. (A) Left column: Uninjected control embryo at stage 39, bilaterally CoMO injected embryo (50 ng/blastomere), mm-FoxO4MO injected embryo (40 ng/blastomere). Right column: FoxO1MO injected embryo (50 ng/blastomere), FoxO6MO injected embryo (30 ng/blastomere), FoxO4MO injected embryo (20 ng/blastomere). All embryos are shown by lateral view. (B) Real time RT-PCR for Sod-2 in wild type and FoxO depleted embryos at stages 18 and 24. FoxO1MO: 35 ng/blastomere, FoxO4MO: 15 ng/blastomere, FoxO6MO: 25 ng/blastomere. (C) FoxO3 depleted embryo and rescue (anterior view). Uninjected control embryo and bilaterally CoMO injected embryo (50 ng/blastomere) at stage 19. Injection of 20 ng FoxO3MO does not interfere with blastopore closure but leads to death during neurulation. Co-injection of 20 ng FoxO3MO together with 500 pg FoxO3 RNA of X. tropicalis can rescue to wild type. (D) Rescue of eye structures in FoxO4 depleted embryos. Magnification of the head at stage 35/36 (lateral view): uninjected control; CoMO injected embryo; FoxO4MO injected embryo, the eye is absent; rescue of the eye by co-injection of 160 pg mutated FoxO4-GR RNA. 15 ng CoMO or FoxO4MO were co-injected with fluorescein in one dorsal blastomere at 4-cell stage. Percentages of phenotypes are given in supplementary Table S4-S6.

response to varying amounts of FoxO4MO are given in Table S7. A comparison of isolated hearts from controls and FoxO4 depleted embryos at stage 43 reveals that looping of heart failed in the absence of FoxO4 (Fig. 6C). Therefore, we performed in situ hybridizations with unilaterally injected embryos for several cardiac marker genes. We observed a lack of myocardin (myocd) expression at the injected side (Fig. 6C) and a severe reduction of its target gene myosin heavy chain α (MHCα) (Fig. 6C), a marker of myocardial differentiation in Xenopus (Small et al., 2005). In contrast, the expression of the

precardiac marker Nkx2.5, which is upstream of myocardin (Ueyama et al., 2003), is not affected (Fig. 6C). BMP4, which maintains Nkx2.5 expression and is necessary for differentiation of cardiac progenitors, (Shi et al., 2000; Walters et al., 2001) is also not affected (data not shown). Moreover, we analyzed unilaterally injected embryos for Tbx20, an early heart marker for myo- and endocardium, and one of its interaction partners, GATA-5 (Stennard et al., 2003). We observed no difference in the expression of both markers by injecting FoxO4MO (Fig. 6C).

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Fig. 6. FoxO4MO injection affects eye, heart and cranial crest development. (A) FoxO4 depletion results in lack of eye structures. Head of an unilaterally FoxO4MO injected embryo (40 ng/blastomere) at stage 39 (lateral view). The eye is almost absent. Middle: Pax-6 whole mount in situ hybridization of control and unilaterally FoxO4MO injected embryos at stage 21. Bottom: Otx-2 whole mount in situ hybridization of control and unilaterally FoxO4MO injected embryos at stage 25. (B) FoxO4 depletion affects cranial crest and jaw development. In situ hybridization of unilaterally FoxO4MO injected embryos with the neural crest markers FoxD3 at stage 23 and AP2α at stage 33. Red arrows indicate the lack of FoxD3 and AP2α expression. Bottom: Alcian blue stained control and unilaterally FoxO4MO injected embryo (30 ng/blastomere) at stage 47. (C) FoxO4 depletion results in failure of heart development. Top: lateral view on control and bilaterally FoxO4MO injected embryo at stage 41. Right: control heart of stage 43 embryo and heart of FoxO4MO injected embryo (20 ng/blastomere at 2-cell stage). Bottom: control- and unilaterally FoxO4Mo injected embryos. myocd and MHCα in situ hybridization reveals reduced expression at the injected side. In situ hybridization of unilaterally FoxO4MO injected embryos with Nkx2.5, GATA-5 and Tbx20. Red asterisks indicate the side of injection. ba, branchial arches; c, conus; ch, ceratohyale; mc, Meckel's cartilage; v, ventricle. Percentages of phenotypes are given in supplementary Table S7.

Aberrant FoxO expression results in reduced proliferation during Xenopus development Since FoxO transcription factors are involved in cell cycle regulation and induce cell cycle arrest, we have investigated cell

proliferation by BrdU assays during X. laevis development in FoxO GOF and LOF embryos at different stages. For gain of function, embryos were analyzed at stage 9.5 and 11.5, respectively. While wild type FoxO3 RNA injection revealed no change of the proliferation rate as compared to uninjected controls, injection of

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FoxO3-TM, FoxO1, FoxO4 and FoxO6 RNA led to a distinct reduction of BrdU positive cells at stage 9.5 (Fig. 7A) and stage 11.5 (data not shown), correlating to a role of FoxO proteins in the suppression of cell proliferation. Surprisingly, we also observe a reduction of BrdU positive cells in FoxO depleted embryos (Figs. 7B–E). However, this reduction becomes apparent only at later stages. As compared to CoMO injected, the number of proliferating cells in FoxO3 depleted embryos does not change before the end of gastrulation (Fig. 7B, left), thereby confirming our previous result that FoxO proteins are dispensable during gastrulation. At stage 15, when most of FoxO3 depleted embryos start to die, a reduction of BrdU positive cells is observed (Fig. 7B, right). Moreover, while most of BrdU positive cells in the controls were found along the neural fold, in FoxO3MO injected embryos the few proliferating cells are dispersed all over the embryo. At stage 28, FoxO1MO and FoxO6MO injected embryos reveal a ubiquitous reduction in the amount of proliferating cells within in the whole embryo (Fig. 7C). This reduction is also observed for bilaterally FoxO4MO injected embryos at stage 30 (data not shown), for unilaterally injected embryos at stage 27 and, interestingly, for distinct areas within the head region of stage 30 embryos, mainly in the branchial arches, the brain and the eye (Figs. 7D–F).

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FoxO proteins affect apoptosis during development of X. laevis Mammalian FOXO proteins trigger programmed cell death in several tissues via the intrinsic or extrinsic apoptotic pathway. They directly induce death genes, like FasL, and are also involved in the transactivation of members of the Bcl-2 family that confer pro- and anti-apoptotic activities and regulate cell survival (Fu and Tindall, 2008). We here investigate the effect of FoxO factors on apoptosis during X. laevis development in gain and loss of function experiments. Cell death in whole embryos is visualized via TUNEL and further specified by measuring caspase activities (Fig. 8). To analyze the gain of function effects we injected daf-16, FoxO1, FoxO3, triple mutated FoxO3 (FoxO3-TM), FoxO4 and FoxO6 RNA into 2-cell stage embryos. At stage 11.5, only few apoptotic cells can be detected in uninjected control embryos, daf-16, FoxO1 and FoxO3 RNA injected embryos (Fig. 8A). Also, injection of FoxO3-TM RNA does not increase the rate of apoptotic cells (data not shown), indicating that even a higher nuclear level of FoxO3 protein does not enhance apoptosis at this stage. In contrast, injection of FoxO6 and, especially, of FoxO4 RNA leads to a distinct increase of apoptotic cells. In order to specify the mechanism, we measured the activities of caspase 8 that is known to trigger the extrinsic apoptotic pathway, of caspase 9, a mediator of the mitochondrial

Fig. 7. BrdU incorporation demonstrates reduced cell proliferation following FoxO overexpression and depletion. (A) Animal view of control embryo, stage 9.5. GOF embryos were bilaterally injected at 2-cell stage with 500 pg/blastomere FoxO1 or FoxO3, 250 pg/blastomere FoxO3-TM, 400 pg/blastomere FoxO4 and 200 pg/blastomere FoxO6 RNA. (B) Bilaterally CoMO and FoxO3MO injected embryos at stage 11.5 (animal view) and stage 15 (dorsal view). (C) CoMO, FoxO1MO and FoxO6MO injected embryo at stage 28 (lateral view). (D) Unilaterally FoxO4MO injected embryo at stage 27. (E) Magnification of the head of an unilaterally FoxO4MO injected embryo at stage 30 (lateral view). Morpholinos were uni- or bilaterally injected at indicated amounts into 2-cell stage embryos. Note that the blue spots represent BrdU positive cells, while the brown color is due to natural pigmentation of embryos. Asterisks indicate the morpholino injected side. ba, branchial arches; e, eye; n, neural fold.

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with the early death of FoxO3 depleted embryos (Fig. 5C). We did not find an increase of apoptosis for FoxO1MO, FoxO4MO and FoxO6MO injected embryos at this stage. At stage 26, FoxO4MO injected embryos show an intense staining in the head and, especially, in the eye. We also checked uni- and bilaterally FoxO4MO injected embryos at stage 31 and detected increased apoptosis in the pharyngeal arches of the injected side (data not shown). Therefore, the TUNEL positive cells correlate with the expression pattern of FoxO4 and, moreover, with the observation of lacking eye and cartilage formation in FoxO4 depletion. At stage 26, FoxO6 depleted embryos also show an increased TUNEL staining for the whole embryo and an intense staining in the head (Fig. 8B). FoxO1MO injected embryos demonstrate a slightly increased number of stained cells at this stage, especially in the head, but less than for FoxO4MO or FoxO6MO. Analysis of caspase activities at stage 19 revealed no increase for FoxO1MO injection, an increased activity of the effector caspases 3/7 for FoxO3 and FoxO4 depletions (3-fold and 4-fold, respectively) and a slight upregulation for FoxO6 depletion. At stage 26, we observed an increase of caspase 3/7 activity subsequent to all FoxOMO injections as compared to uninjected controls or CoMO injected embryos (Fig. 8B). Discussion FoxO genes show partially overlapping expression patterns

Fig. 8. FoxO proteins affect apoptosis. (A) Embryos were injected at 2-cell stage with the same amounts as given in Fig. 7. At stage 11.5, embryos were analyzed for apoptosis by TUNEL (animal view) and for caspase activities using caspase 8, 9 and 3/7 Glo assays. (B) Embryos injected with FoxO3MO and corresponding controls were analyzed by TUNEL at stage 15 (dorsal view, left), embryos injected with FoxO1MO, FoxO4MO or FoxO6MO were analyzed at stage 26 (lateral view, bottom). Caspase 8, 9 and 3/7 activities were determined at stages 19 and 26, respectively.

apoptotic pathway, and of the effector caspases 3/7. For all injections we did not find any significant changes of caspase 8 and caspase 9 activities, whereas effector caspases 3/7 are upregulated. FoxO6 overexpression leads to a 2-fold increase, while the activity in FoxO4 injected embryo is 4fold increased in comparison to uninjected controls. We next examined, whether loss of FoxO function affects the number of apoptotic cells or caspase activities (Fig. 8B). Therefore, we injected FoxO antisense morpholino oligonucleotides and analyzed the embryos at different developmental stages. Almost no apoptotic cells or changes of caspase activities were detected at stage 11 (data not shown). However, at stage 15, FoxO3 depleted embryos show a high number of apoptotic cells being scattered over the whole embryo (Fig. 8B). This observation correlates

In addition to the already known FoxO1 and FoxO3 genes (Pohl et al., 2004) we have now identified the FoxO4 and FoxO6 genes in Xenopus. The semi-quantitative RT-PCR analysis of adult tissues revealed that expression of some FoxO genes is highly enriched in specific tissues, like the FoxO6 transcripts, which are mainly localized in brain and eye. This tissue specificity has also been shown for the murine Foxo genes by Northern blot analysis (Furuyama et al., 2000). While murine Foxo1 was shown highly expressed in brown and white adipocyte tissue but only weakly in heart, Foxo3 is almost absent from adipocytes but strongly expressed in heart. However, we also observe an overlap of expression in many examined organs. Analyzing the temporal expression patterns of FoxO genes, we observe that FoxO3 has a strong maternal contribution, whereas FoxO1, FoxO6 and also FoxO4 seem to be progressively upregulated after the onset of zygotic transcription and should therefore be involved in cellular differentiation processes subsequent to midblastula transition. Spatial expression of FoxO4 in neurulating embryos is predominantly observed in neural crest cells and in the early eye field, while at later stages we find expression in the branchial arches, the head mesenchyme, the brain, the pronephric duct and in the presumptive retina. FoxO6 was mainly found in the hatching gland and branchial arches. FoxO1 expression was previously reported in the pronephros, within head mesenchyme in front of the eye, within branchial arches and in the liver primordium, while FoxO3 becomes apparent at late neurula within the anterior neural plate and in neural crest cells. Later, expression of FoxO3 is observed in a variety of organs and tissues, like eye, branchial arches and somites (Pohl et al., 2004). Noteworthy, there is some similarity between the patterns of FoxO1 and FoxO4 on the one side and a rather strong similarity between FoxO3 and FoxO6 on the other side. This partial overlap is also reflected by a closer sequence relationship between FoxO1 and FoxO4 as well as of FoxO3 and FoxO6 (see Fig. 1D). This gives rise to the speculation, that the ancestral FoxO gene was primarily duplicated to two genes representing FoxO1/4 and FoxO3/6 precursors, which later on were duplicated to presently existing genes. Conservation of regulatory elements within promoter sequences should be responsible for observed similarities of expression patterns. FoxO overexpression inhibits gastrulation and directs cells into primitive ectodermal fate Overexpression of FoxO1, FoxO4, FoxO6 and the C. elegans homologue daf-16 revealed severe gastrulation defects resulting in

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open or even lacking blastopore. This phenotype was accompanied by a downregulation of mesodermal and endodermal marker genes. The levels of transcripts for neuroectodermal and neural markers are also decreased. In contrast, Xema (Xenopus ectodermally expressed mesendoderm antagonist; Suri et al., 2005) is strongly activated. Furthermore, we observed a slight activation of Grhl1, a transcription factor that was shown to be essential for ectodermal ontogeny and necessary for expression of epidermal genes (Tao et al., 2005). Upregulation of epidermal cadherin and epidermal keratin, indicating epidermal differentiation, can only be observed for FoxO1 injection, suggesting functional differences between the FoxO genes. Moreover, it is known that FoxO genes are involved in the regulation of apoptosis. We found high levels of apoptosis in FoxO4 RNA injected embryos, and we cannot exclude that failure in gastrulation is due to an increased death of certain cell types. However, we observed almost no apoptosis in FoxO1 RNA injected embryos and only a slight increase in the animal hemisphere following FoxO6 RNA injection. Furthermore, we found decreased numbers of proliferating cells. This finding is in correlation to previous studies, which show that Foxo proteins inhibit the cell cycle. They stimulate expression of the cyclindependent kinase inhibitor p21WAF1 to induce G1-phase arrest of the cell cycle (Seoane et al., 2004) and they also up regulate the cyclindependent kinase inhibitor p27KIP1 (Nakamura et al., 2000). Moreover, growth arrest and DNA damage-inducible protein 45 alpha (Gadd45α) was shown to be a direct target of FoxO3a (Tran et al., 2002). Our findings suggest that the failure of gastrulation in FoxO GOF embryos is due to a decrease of cell proliferation and an increase of cell death. The observed increase of Xema and Grhl1 in FoxO RNA injected embryos demonstrates that FoxO overexpression can force cells into a primitive ectodermal cell fate but inhibits proper differentiation into neural or epidermal cell lineage. Therefore, our results give rise to the postulate that only low or even absent FoxO expression is a pre-requisite for normal gastrulation. FoxO function is regulated by several posttranslational modifications Various posttranslational modifications of mammalian FOXO proteins have been reported. The first identified modification being even conserved in the nematode C. elegans was the phosphorylation by the PI3K/Akt signaling pathway. This modification is achieved by insulin/IGF-1 and several other factors leading to nuclear export and functional inactivation of FoxO proteins (Ogg et al., 1997; Brunet et al., 1999; Kops et al., 1999). Three Akt-1 phosphorylation sites were found in each mammalian FOXO1, FOXO3 and FOXO4 proteins. It is generally accepted that phosphorylation via Akt-1 removes FOXO proteins from the nucleus to the cytosol (Huang and Tindall, 2007). Interestingly, FoxO6 lacking the third Akt phosphorylation motif shows predominant nuclear localization (Jacobs et al., 2003), suggesting that this site is crucial for the shuttle process (van der Heide et al., 2005). This is also shown for the FoxO6 protein from Xenopus (see Fig. 4S). We observed differences in the intensities of the gastrulation phenotypes gained by FoxO overexpression. In contrast to the other FoxO genes, FoxO3 overexpression had no influence on gastrulation, the phenotype of FoxO1 overexpression was weak, while FoxO6 and FoxO4 resulted in severe gastrulation defects. This observation is unlikely due to different translation efficiencies, as in vitro translation assays led to faithful synthesis of all FoxO proteins at similar quantities (data not shown). However, the gastrulation phenotype was obtained by injecting the triple mutated FoxO3 construct, where the putative Akt responsive serine and threonine residues were changed to alanine. This experiment indicates that differences in protein function of FoxOs might be caused by posttranslational modifications. The intensity of the GOF FoxO6 phenotype can be interpreted by the missing third Akt-1 phosphorylation site. With regard to the intensity of the FoxO4

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phenotype, we suppose that this might be due to the additional, putative JNK phosphorylation site, which is only found in FoxO4/O6 proteins. JNK phosphorylates Foxo4 and, in contrast to Akt-1, triggers its relocalization from the cytosol to the nucleus. Moreover, JNK also phosphorylates the 14-3-3 proteins, resulting in a release of FoxO (Calnan and Brunet, 2008). The observation, that even high doses of FoxO3 RNA do not result in a phenotype, whereas FoxO3-TM induces severe gastrulation defects and a reduction in cell proliferation, suggests that Akt phosphorylation is the major modification mechanisms for the inactivation of FoxO3. We also demonstrate differences between the FoxO genes in their capability to induce apoptosis. Although upregulations of proapoptotic genes were shown for the mammalian FOXO1/FOXO3 and FOXO4 genes (Barreyro et al., 2007; Tang et al., 2002), we only observed a strong stimulation of apoptosis in FoxO4 injected embryos. Injections of FoxO1 or FoxO3 RNA show almost no and FoxO6 only a slight increase of apoptosis. Interestingly, the constitutive active FoxO3-TM RNA did not lead to a severe increase of apoptosis by transcriptional activation of pro-apoptotic genes as it was reported for primary T-cells (Stahl et al., 2002). Therefore, FoxO factors show functional differences in the regulation of pro-apoptotic genes in Xenopus development. FoxO4 is necessary for eye, heart and craniofacial development The loss of function experiments of FoxO1, FoxO4 and FoxO6 led to multiple defects in organogenesis, retardation of development and to crippled embryos. Embryos injected with FoxO3MO did not even pass normal neurulation. Moreover, we observed increased rates of apoptosis coupled with a decrease of proliferating cells after loss of function. This indicates some profound and ubiquitous effects by FoxO depletion on embryogenesis. One aspect might be the involvement of FoxO factors in cellular metabolism, another one the prevention of oxidative stress. It was shown that Sod-2, being involved in reduction of peroxides, is positively regulated by FoxO factors (Honda and Honda 1999; Yanase et al., 2002). Indeed, we observed a dramatic decrease of Sod-2 expression in FoxOMO injected embryos (Fig. 5). This finding might explain the whole-body wasting phenotype as well as the lack of proliferation and increased cell death. It is known that O2-, which is produced in the mitochondria by the electron transport system, is detoxified by Sod-2 and a reduction or lack of Sod-2 activity induces apoptosis (Wallace, 2001). Reduced cell proliferation is also generated by cell damage induced cell cycle arrest. Since Foxo4 (−/−) mice develop normally and cannot be distinguished from wild type mice, the Foxo4 function during early development remained unclear. FoxO4 depletion in Xenopus results in lack of eye, cartilage defects and malformation of the heart. Inhibition of FoxO4 reduces the early eye field marker Pax-6 and Otx-2. Disruption or blocking the paired box transcription factor Pax-6 in mice as well as in frogs results either in absence or in small and abnormally formed eyes (Grindley et al., 1995; Chow et al., 1999). The eye field originates from the forebrain. Deficiency in Otx-2 signaling results in lack of eye structure (Acampora et al., 1995). Since we find a severe decrease of Otx-2 in the forebrain, it seems that FoxO4 is necessary for proper eye formation already at forebrain development. It should be noted that these observations might be in conflict with recent reports on the role of IGF. IGF RNA injection has been shown to induce ectopic eyes and an expansion of anterior neural tissues (Pera et al., 2001). Since IGF activates Akt-1 that, in turn, inactivates FoxO, IGF should antagonize FoxO. This is further supported by our findings, that gastrulation can be restored in FoxO GOF embryos by injecting components of the IGF pathway (IGF-1/IGF-2/Akt-1, see Fig. 4), However, injection of Akt-1 RNA, being downstream of IGF but directly upstream of FoxO, cannot expand anterior neural tissues (Fig. S2B). This failure is also not due to lacking activation of Akt-1 by phosphorylation of S473 and T308 in response to IGF signaling. We

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mutated these sites (T309 and S474) to glutamic acid (the corresponding mutant is designated as Akt1/double mutated: AktDM) to mimic the effect of phosphorylation and to enforce the effect of Akt-1 (Alessi et al., 1996; Bellacosa et al., 1998). Neither injection of Akt-1 nor of Akt-DM can expand the Otx-2 expression domain as reported for IGF injection (Pera et al., 2001). These data clearly demonstrate that the neural- and head inducing effect of IGF is not mediated by the Akt/FoxO pathway, but by a parallel signal cascade downstream of IGF. Therefore, the reduction of Otx-2 and Pax-6 expression in FoxO4 depleted embryos cannot simply be correlated to IGF signaling effects. We also found a loss of cranial cartilage in FoxO4 depleted embryos. It is known that the cranial cartilage originates from the anterior neural crest cells. The neural crest cells originally delaminate from the neural tube, migrate lateral of the neural tube and differentiate in multiple cell types (Dottori et al., 2001). As FoxO4 is expressed in the neural crest, we investigated the expression pattern of FoxD3 and AP2α in FoxO4MO injected embryos. FoxD3 plays a role in neural crest cell migration (Pohl and Knöchel, 2001) and AP2α is required for neural crest induction (Luo et al., 2003). Both markers are no longer expressed in the ventrally migrating cell population. We actually do not know, whether absence of staining is due to misleading of migrating crest cells, changes in cell fate or in cell death. However, lack of ventral FoxD3 and AP2α expression indicates that migration of cranial crest cells is affected. In addition, TUNEL studies revealed a high rate of apoptotic cells within the FoxO4 expression domains including the branchial arches. This observation indicates that a lack of FoxO4 signaling directs cells to apoptosis. We also observed intense staining of apoptotic cells in the brain and the eye field, suggesting that cells of the eye placode undergoes programmed cell death. The fact that FoxO4 injection also increases apoptosis in gain of function experiments should not be taken as discrepancy. While we suppose that FoxO4 directly activates proapoptotic genes, as it was shown for the mammalian homologue (Lee et al., 2008; Tang et al., 2002), the increase of apoptosis in FoxO depleted embryos might be caused by a lack of signaling. Thereby, cells might be kept in a pre-differentiated state and undergo programmed cell death. Moreover we found a reduction of BrdU positive cells in the branchial arches and in the eye, which can be an additional reason for the lack of these structures. Finally, the loss of FoxO4 function also leads to deficiencies in heart formation. Although no intense FoxO4 staining was visible in the embryonic heart by whole mount in situ hybridization, we found by semi-quantitative RT-PCR that FoxO4 is strongly expressed in adult heart. This finding is consistent with previous data from mice, where strongest expression of Foxo4 was found in skeletal muscle and in heart (Furuyama et al., 2000). Mammalian FoxO genes have been reported to play a diverse role in the heart, like for example regulation of oxidative stress or of cell cycle. We found a lack of myocardin (myocd) expression in FoxO4 depleted embryos. Myocardin is a smooth and cardiac muscle specific transcriptional cofactor of the serum response factor (SRF), which is essential for cardiac differentiation (Wang et al., 2001). Myocardin loss of function experiments in Xenopus revealed that this factor is essential for expression of cardiac differentiation markers and heart tube formation, while expression of the precardiac marker Nkx2.5 remains unaffected (Small et al., 2005). In support of these findings, we observe a downregulation of MHCα in FoxO4 depleted embryos, whereas Nkx2.5, GATA-5, Tbx-20 and BMP4 transcripts are not decreased, indicating that the heart defect is due to a misregulation of myocd. The mammalian myocd promoter contains an upstream enhancer element with several Foxo binding sites. This enhancer was shown to be stimulated via Foxo4 (Creemers et al., 2006). Whether the upstream region of the Xenopus myocd gene also contains an enhancer with FoxO binding sites, as reported for other organisms (Creemers et al., 2006), is under current investigation.

Acknowledgments We thank Nicole Heymann and Brigitte Korte for skilful technical assistance. We are indebted to C. Kenyon, P. A. Krieg and E.M. Pera for gifts of plasmids. This work was supported by grants from Novartis to W.K. and from the Deutsche Forschungsgemeinschaft (SFB497/A3 to W.K. and SFB497/B9 to F.O.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ydbio.2009.10.036. References Acampora, D., Mazan, S., Lallemand, Y., Avantaggiato, V., Maury, M., Simeone, A., Brûlet, P., 1995. Forebrain and midbrain regions are deleted in Otx2-/- mutants due to a defective anterior neuroectoderm specification during gastrulation. Mech. Dev. 51, 83–98. Alessi, D.R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., Hemmings, B.A., 1996. EMBO J. 15, 6541–6551. Andersen, C.B., Sakaue, H., Nedachi, T., Kovacina, K.S., Clayberger, C., Conti, M., Roth, R.A., 2003. 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