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Klf5 Is Involved in Self-Renewal of Mouse Embryonic Stem Cells

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Klf5 Is Involved in Self-Renewal of Mouse Embryonic Stem Cells Short Report 2629 Klf5 is involved in self-renewal of mouse embryonic stem cells Silvia Parisi1,2,*, Fabiana Passaro1,3,*, Luigi Aloia1,2, Ichiro Manabe4, Ryozo Nagai5, Lucio Pastore1,3 and Tommaso Russo1,3,‡ 1CEINGE Biotecnologie Avanzate, 80145 Napoli, Italy 2European School of Molecular Medicine, SEMM, 80145 Napoli, Italy 3Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy 4Nano-Bioengineering Education Program and 5Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected]) Accepted 15 May 2008 Journal of Cell Science 121, 2629-2634 Published by The Company of Biologists 2008 doi:10.1242/jcs.027599 Summary Self-renewal of embryonic stem cells (ESCs) is maintained by undifferentiated state by Klf5 is, at least in part, due to the a complex regulatory mechanism involving transcription factors control of Nanog and Oct3/4 transcription, because Klf5 directly Oct3/4 (Pou5f1), Nanog and Sox2. Here, we report that Klf5, a binds to the promoters of these genes and regulates their Zn-finger transcription factor of the Kruppel-like family, is transcription. involved in ESC self-renewal. Klf5 is expressed in mouse ESCs, blastocysts and primordial germ cells, and its knockdown by RNA interference alters the molecular phenotype of ESCs, Supplementary material available online at thereby preventing their correct differentiation. The ability of http://jcs.biologists.org/cgi/content/full/121/16/2629/DC1 Klf5 to maintain ESCs in the undifferentiated state is supported by the finding that differentiation of ESCs is prevented Key words: Differentiation, Kruppel-like factors, Nanog, Oct3/4, when Klf5 is constitutively expressed. Maintenance of the Self-renewal Introduction Results and Discussion ESCs derived from the inner cell mass of the blastocyst can Klf5 is expressed in mouse ESCs and its knockdown Journal of Cell Science differentiate into primitive ectoderm, primitive endoderm and suppresses normal ESC differentiation trophoectoderm cells, and in turn into all cell types present in the The screening of a collection of short hairpin RNAs (shRNAs) embryo. ESCs are maintained in the undifferentiated state during designed to target mouse mRNAs allowed us to observe that the self-renewal by a complex regulatory network involving three shRNA targeting Klf5 mRNA was able to interfere with ESC transcription factors, namely Oct3/4 (Pou5f1) (Nichols et al., 1998), differentiation (see supplementary material Fig. S1). Klf5, also Nanog (Chambers et al., 2003; Mitsui et al., 2003) and Sox2 (Avilion known as intestinal-enriched factor and basic transcription et al., 2003). These factors regulate their own expression and that element binding protein 2, is a Zn-finger transcription factor of many other genes (Boyer et al., 2005; Loh et al., 2006). Other belonging to the Sp/Kruppel-like family. In adults, it is expressed transcription factors play important roles in ESC pluripotency and in the proliferating crypt cells of the intestinal epithelium and at self-renewal (Niwa, 2007). These data illustrate the complexity of low levels in the testis, uterus, placenta, lung and in the proliferating basal layer of the epidermis (Ohnishi et al., 2000). transcription regulation in ESCs, which is still not completely Klf5 knockout causes early embryonic lethality (Shindo et al., understood. 2002), which suggests that this factor plays a key role during Recently, several results indicate that transcription factors early development. belonging to the Kruppel-like family could have an important role Klf5 mRNA and protein are present in undifferentiated ESCs in the regulation of ESCs. Ectopic expression of Klf4, together and levels rapidly decrease after induction of ESC differentiation with Oct3/4, Sox2 and Myc, results in the conversion of by two different approaches (Fig. 1A,B). Immunostaining differentiated cells into pluripotent ES-like cells (Takahashi et al., demonstrated that Klf5 is expressed at various levels in almost all 2006; Okita et al., 2007; Werning et al., 2007; Maherali et al., undifferentiated ESCs (Fig. 1C) and that it substantially colocalises 2007). Recent findings indicate that Klf2 or Klf5 can replace Klf4 with Oct3/4 and Nanog [964 out of 1100 cells (87.6%) were positive in the gene combination inducing cell reprogramming (Nakagawa for both Oct3/4 and Klf5; 512 out of 678 cells (75.5%) were Nanog et al., 2008) and that triple knockdown (KD) of Klf2, Klf4 and and Klf5 positive; 625 out of 720 cells (86.8%) expressed both Klf5 abolishes the undifferentiated phenotype of ESCs (Jiang et Oct3/4 and Nanog]. After 3 days in differentiation conditions, the al., 2008). Klf5 signal disappeared from most cells, as observed for Oct3/4 In this study, we demonstrate that KD of even only Klf5 and Nanog (supplementary material Fig. S2). The expression of abolishes the ESC undifferentiated phenotype, whereas its Klf5 mRNA in vivo is in agreement with that observed during in constitutive expression prevents ESC differentiation. vitro differentiation of ESCs. In fact, we found the Klf5 transcript 2630 Journal of Cell Science 121 (16) in blastocysts at embryonic day (E)3.5 but not in the epiblast of E6.5 embryos (Fig. 1D). Immunostaining of pre-implantation embryos revealed nuclear Klf5 in the morula (Fig. 1E) and in many cells of the blastocyst at Theiler stage 4 (Fig. 1E,F), when the blastocoelic cavity is formed. At these stages, the cells also expressed Oct3/4 and Nanog. At a later stage, the expression of Klf5 persisted (supplementary material Fig. S2). Immunostaining of sections from E11.5 embryos revealed that Klf5 is also expressed in small groups of cells in the genital ridge, coincident with primordial germ cells expressing Oct3/4 (Fig. 1H). ESCs lose their undifferentiated phenotype as a consequence of Klf5 KD The observation that Klf5 expression is restricted to undifferentiated ESCs both in vitro and in vivo and is tightly regulated when differentiation occurs suggests that it might be required to maintain ESCs in an undifferentiated state. To address this hypothesis, we explored the effects of Klf5 KD in undifferentiated ESCs. To this aim, ESCs were transfected with the previously used Klf5 shRNA or with a mixture of four short interfering RNAs (siRNAs), all targeting different regions of the Klf5 mRNA. Whereas non-silencing (CRL)- shRNA-transfected cells were indistinguishable from untransfected cells, Klf5 KD resulted in the appearance of clusters of enlarged flattened cells (supplementary material Fig. S3). To verify that these morphological changes result from loss of the undifferentiated phenotype, we counted alkaline-phosphatase-positive colonies (APcs), as a marker of undifferentiated ESCs, in cells plated at clonal density (~80 cells/cm2). Although grown in the presence Journal of Cell Science of leukaemia inhibitory factor (LIF) and serum, Klf5- KD cells lost their undifferentiated phenotype, as witnessed by the drastic reduction of APcs (Fig. 2A). Accordingly, Oct3/4 and Nanog expression was significantly decreased, as demonstrated by immunostaining (Fig. 2B) and reverse-transcriptase (RT)-PCR (Fig. 2C). Fig. 1. Klf5 expression in ESCs. (A,B) Klf5 mRNA (A) and protein (B) levels decrease soon after the induction of ESC differentiation. ESCs were differentiated as embryoid Furthermore, Oct3/4 KD decreased levels of the bodies (EBs) or in monolayer. (C) Klf5 is highly expressed in undifferentiated ESCs and cognate mRNA and protein by >50% and was colocalised with Oct3/4 and Nanog. Scale bars: 50 μm. (D) RT-PCR of Klf5 in ESCs, accompanied by a significant reduction of Sox2 and and in E3.5 and E6.5 embryos. (E) Klf5 is expressed in morulae together with Oct3/4 Nanog mRNA levels. In these conditions, Klf5 mRNA and Nanog. (F,G) E3.5 embryos express Klf5, Oct3/4 and Nanog in the inner cell mass. μ was significantly decreased (Fig. 2D), which reinforces Scale bars: 20 m. (H) Two consecutive sections of an E11.5 embryo were stained with anti-Klf5 and -Oct3/4 antibodies. The arrowheads indicate the groups of cells in the the concept that the expression of Klf5 is restricted to the genital ridge (primordial germ cells) expressing both Klf5 and Oct3/4. The arrow ESC undifferentiated state. indicates the stomach epithelium, where Klf5 is also expressed (Ohnishi et al., 2000). To explore phenotypic changes induced by Klf5 KD, we analysed several markers of cell fate. As shown in Fig. 2E, markers of endoderm (Gata4, Hnf4 and Sox17), ectoderm Klf5 constitutive expression prevents ESC differentiation (Sox1) and visceral endoderm (Afp) were undetectable in both We then examined ESC clones stably expressing Klf5 under the Klf5- and CRL-shRNA-transfected cells; mRNAs for the control of a constitutive β-actin promoter (see supplementary mesoderm markers brachyury and Meox1, and for the trophoblast material Fig. S5). Mock-transfected ESC clones plated at ~80 markers Cdx2, Eomes and PL-1, appeared only in Klf5 KD cells. cells/cm2 showed only a few APcs (60±3.8 APcs/100-mm plate, Immunostaining of Klf5 KD cells showed that 12.3±0.2% of cells n=4) 6 days after LIF withdrawal, whereas, despite LIF withdrawal, (n=920) expressed brachyury and 11.2±0.6% (n=950) expressed ESCs stably expressing exogenous Klf5 showed 912.5±33 Cdx2 (supplementary material Fig. S4). The remaining cells did APcs/100-mm plate (n=4, Fig. 3A). As shown in Fig. 3B-D, after not express any examined differentiation markers, and some of 5 days in differentiation conditions, exogenous Klf5 expression these cells still expressed Nanog (28.5±3%) and/or Oct3/4 sustains Oct3/4 and Nanog expression whereas, in mock-transfected (37.8±3.5%). cells, Oct3/4 and Nanog expression is strongly decreased. Klf5 in self-renewal of ESCs 2631 immunoprecipitation (ChIP) experiments, which demonstrated the direct interaction of Klf5 with the Nanog promoter (Fig.
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