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First published online 24 July 2008
doi: 10.1242/jcs.027599

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Klf5 is involved in self-renewal of mouse embryonic stem cells

Silvia Parisi^1,2,*, Fabiana Passaro^1,3,*, Luigi Aloia^1,2, Ichiro Manabe⁴, Ryozo Nagai⁵, Lucio Pastore^1,3 and Tommaso Russo^1,3,

¹ CEINGE Biotecnologie Avanzate, 80145 Napoli, Italy
² European School of Molecular Medicine, SEMM, 80145 Napoli, Italy
³ Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy
⁴ Nano-Bioengineering Education Program, The University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan
⁵ Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan

View larger version (44K): [in this window] [in a new window]   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 bodies (EBs) or in monolayer. (C) Klf5 is highly expressed in undifferentiated ESCs and colocalised with Oct3/4 and Nanog. Scale bars: 50 µm. (D) RT-PCR of Klf5 in ESCs, and in E3.5 and E6.5 embryos. (E) Klf5 is expressed in morulae together with Oct3/4 and Nanog. (F,G) E3.5 embryos express Klf5, Oct3/4 and Nanog in the inner cell mass. Scale bars: 20 µm. (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 genital ridge (primordial germ cells) expressing both Klf5 and Oct3/4. The arrow indicates the stomach epithelium, where Klf5 is also expressed (Ohnishi et al., 2000).

View larger version (52K): [in this window] [in a new window]   Fig. 2. Klf5 is required to maintain ESCs in an undifferentiated state. (A) Klf5 KD causes a decrease in the number of APcs. CRL- or Klf5-shRNA transfected ESCs were plated at 80 cells/cm2 and grown for 6 days. The number of APcs was 2040±55 per 100-mm plate (n=5) for CRL shRNA (upper panel) and 158±14 per 100-mm plate (n=5) for Klf5 shRNA (lower panel). Scale bar: 1 mm. (B) ESCs shown in A were stained with anti-Oct3/4 or -Nanog antibodies. In Klf5-KD cells, the expression of these two proteins is drastically decreased. Scale bars: 100 µm. (C) Klf5 KD is accompanied by a significant (P<0.01) decrease of Oct3/4, Sox2 and Nanog mRNAs. β-actin mRNA was used as a control. Standard errors of three independent experiments are reported. (D) Silencing of Oct3/4 downregulates Klf5 expression. (E) RT-PCR analysis of mRNAs shows that Klf5 KD causes illegitimate expression of brachyury and Meox (mesoderm), and of Cdx2, PL-1 and Eomes (trophoblast).

View larger version (56K): [in this window] [in a new window]   Fig. 3. Klf5 constitutive expression prevents ESC differentiation. (A) ESCs that were mock-transfected (right panel) or transfected with Klf5 expression vector (left panel) were plated at low density (80 cells/cm2) and grown for 6 days without LIF. Scale bars: 1 mm. (B,C) Mock- or Klf5-transfected ESCs were grown for 5 days in differentiation conditions and stained with anti-Nanog and -Oct3/4 (B) or -Klf5 and -Nanog (C) antibodies. Scale bars: 100 µm. (D) Western blot analyses of Oct3/4 and Nanog in undifferentiated and differentiated ESCs. Protein extracts are from four clones stably expressing Klf5-overexpressed (Klf5 ov.) and one from mock-transfected cells.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Klf5 is a component of the Oct3/4 and Nanog transcription-factor network. (A) Nanog-Luc or Pou5f1-Luc were co-transfected with Klf5 (Klf5 ov.), Klf5 shRNA or control (mock) vectors. (B) 3xFLAG-Klf5 transfected ESCs were processed for ChIP with antibody for the FLAG epitope or IgG as control. The amount of precipitated DNA was calculated relative to the total input chromatin. (C) The indicated oligonucleotide, designed on the basis of the Nanog promoter (Wu and Yao, 2005), was incubated with nuclear extracts of mock-transfected (lanes 2-4) or 3xFLAG-Klf5-transfected (lanes 6-9, 11,12) cells. Nuclear extracts were preincubated with 50-fold molar excess of unlabelled oligo probe (lanes 3 and 7) or unrelated oligo (lanes 4 and 8), or mutant oligo in which the sequence –78 to –72 (GGGTGGG, underlined) was changed into AGATAGA to disrupt the candidate cis-element of Klf5. In lane 12, nuclear extracts were preincubated with anti-Klf5 antibody. Relevant bands are indicated (arrows) as follows: (a) candidate band for endogenous Klf5; (b) specific shifted band not related to Klf5; (c) additional band due to exogenous 3xFLAG-Klf5. (D) ChIP experiments with anti-Nanog antibody on the Klf5 promoter. K1 and K2 indicate two oligonucleotide pairs that amplify the regions of the Klf5 gene (supplementary material Fig. S7). The differences with control antibody and control DNA were significant (P<0.001). (E) Real-time PCR of Klf5, Nanog, Oct3/4 and Sox2 mRNAs in Klf4-KD or -overexpressing cells. *P<0.001, **P<0.01. (F) mRNA levels of Klf2 and Klf4 in ESCs transfected with Oct3/4, Nanog or Klf5 shRNAs or with Klf5 overexpressed (Klf5 ov). *P<0.001.

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