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First published online 16 October 2007
doi: 10.1242/jcs.007856

Journal of Cell Science 120, 3859-3869 (2007)
Published by The Company of Biologists 2007
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Sox17 plays a substantial role in late-stage differentiation of the extraembryonic endoderm in vitro

Masafumi Shimoda1,2, Masami Kanai-Azuma3, Kenshiro Hara4, Satsuki Miyazaki2, Yoshiakira Kanai4, Morito Monden1 and Jun-ichi Miyazaki2,*

1 Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2 Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
3 Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan
4 Department of Veterinary Anatomy, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan


Figure 1
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  		Fig. 1. Generation of ESCs with inducible exogenous Sox17 expression. (A) Schematic representation of the knock-in strategy of the inducible Sox17 expression cassette into the ROSA26 (R26) locus. The inducible Sox17 expression cassette was inserted into the XbaI site in the ROSA26 first intron. This cassette consisted of a splice acceptor (SA), floxed promoter-less neomycin-resistance gene (neo), tTA, insulator derived from the chicken beta-globin locus, tTA-responsive human cytomegalovirus minimal promoter (hCMV*1), rabbit beta-globin second intron, mouse full-length Sox17 cDNA, internal ribosome entry site (IRES) and EGFP cDNA. Diphtheria toxin A (DTA) cDNA driven by the mouse phosphoglycerate kinase 1 promoter (pPGK) was placed at the 3' end of the targeting construct for negative selection. After the targeting of the ROSA26 locus, neo was excised by the transient expression of Cre recombinase. The locations of the PCR primers used to detect the exogenous Sox17 transcript (primer F and primer R) are indicated. (B) RT-PCR analysis of exogenous Sox17 expression in ESCs grown in the presence or absence of Tet. The black arrowhead denotes the predicted size of the RT-PCR product (2.1 kb). Notice that this size is 0.5 kb less than that of the product derived from genomic DNA as a template (gray arrowhead). Actb is shown as the loading control. (C) Western blotting of the Sox17 protein. The cell lysates of ESCs grown with or without Tet were loaded.

 

Figure 2
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  		Fig. 2. Effect of forced Sox17 expression on ESC differentiation. (A) Morphology and EGFP fluorescence of ESCs grown with or without Tet. Bar, 100 µm. (B) RT-PCR analysis of ExE markers in the ESCs grown with or without Tet. cDNA derived from EBs of parental ESCs grown without LIF for various time periods was used as a positive control, and the corresponding RNA was used as the RT control. The Sox17 primer set was designed to detect both endogenous and exogenous Sox17. (C) Morphology and EGFP fluorescence of EBs. ESCs grown with or without Tet were allowed to aggregate and were grown in LIF-deficient medium with the same Tet status for 6 days. Bar, 200 µm. (D) RT-PCR analysis of germ-layer markers in EBs grown with or without Tet in LIF-deficient medium. Two independent targeted clones were examined with similar results (A-D).

 

Figure 3
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  		Fig. 3. Morphology of EBs grown with or without Tet in LIF-supplemented medium. After culture in LIF-supplemented medium without Tet for induction of exogenous Sox17, the ESCs were allowed to aggregate and were grown in the same medium for 10 days (Tet –). Control EBs were prepared using the same procedure, except that Tet was constantly supplemented to the medium (Tet +). (A,B) Bright-field images. Bars, 500 µm. (C-F) Toluidine-blue staining. Bars, 50 µm (C,D) and 10 µm (E,F). (G-K) TEM images. Boundaries between the PrE-like surface cells and the subjacent cells are shown as broken lines (G,I). The asterisks show the basement membrane-like structure (H,K). Arrows indicate vacuoles (J). Bars, 10 µm (G,H), 2 µm (I,K) and 1 µm (J). (L,M) PAS staining. The inset shows a magnified image (L). Bars, 100 µm. (N,O) Phase-contrast images of EBs grown in gelatin-coated tissue-culture dishes for 1 day. Bars, 300 µm. Two independent clones were examined with similar results (A-O).

 

Figure 4
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  		Fig. 4. Immunostaining of EBs with ExE markers. Anti-COUP-TF1 (A), anti-Gata4 (B), anti-Krt19 (C) and anti-Foxa2 (D) antibodies were used for immunostaining of EBs grown with (+) or without (–) Tet in LIF-supplemented medium. Two independent clones gave similar results. Bars, 100 µm.

 

Figure 5
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  		Fig. 5. The temporal expression pattern of ExE markers during EB differentiation. After culture in LIF-supplemented medium with or without Tet for several passages, ESCs with the inducible Sox17 transgene were allowed to aggregate and were grown in the same medium for the indicated periods. Additionally, the parental ESCs were allowed to aggregate and were grown with or without LIF for the indicated periods. The expression levels relative to those in EBs derived from parental ESCs grown for 6 days are shown as the mean values of duplicate experiments. The Sox17 primer set was designed to detect both endogenous and exogenous Sox17.

 

Figure 6
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  		Fig. 6. The impact of Nanog misexpression on ExE differentiation promoted by forced Sox17 expression. ESCs bearing the inducible Sox17 transgene were stably transfected with an empty vector or a constitutive Nanog expression vector. After being cultured in the absence of Tet in LIF-supplemented medium for Sox17 induction, the ESCs were allowed to aggregate and were grown in the same medium for 10 days. (A,B) Bright-field (A) and fluorescence (B) images of EBs derived from a control (Tet –, Nanog –) clone and a Nanog-misexpressing (Tet –, Nanog +) clone. Bars, 200 µm. (C,D,E) Immunostaining of the control and Nanog-misexpressing EBs with anti-Nanog (C), anti-Gata4 (D) and anti-Krt19 (E) antibodies. Bars, 50 µm. Three independent Nanog clones and two independent control clones were examined with similar results (A-E). (F) Expression of ExE markers in the control (white bars) and Nanog-misexpressing (black bars) EBs. The expression levels relative to those in the control EBs, measured by qRT-PCR, are shown as the mean ± s.d. values of three independent experiments.

 

Figure 7
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  		Fig. 7. Effect of Sox17 deficiency on ExE differentiation. (A) The temporal expression pattern of ExE markers in wild-type (WT) and Sox17–/– (KO) EBs. RNA from EBs grown without LIF for the indicated periods was subjected to qRT-PCR. The expression levels relative to the maximal levels are shown as the mean±s.d. values of two independent experiments. (B) Toluidine-blue staining of EBs grown without LIF for 3 or 9 days. Arrows, PrE-like cells; arrowheads, PE-like cells. Bars, 10 µm. (C) Immunostaining for COUP-TF1 and Gata4 in EBs grown for 4, 9 or 12 days. Bars, 50 µm.

 

Figure 8
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  		Fig. 8. Expression of Sox7 and Sox17 in Sox17 mutant extraembryonic tissues at E7.75-E8.0. The amounts of Sox7 and Sox17 transcripts relative to those in wild-type (+/+) tissues, measured by qRT-PCR, are shown as the mean ± s.d.

 

Figure 9
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  		Fig. 9. Model of the regulation of differentiation of ESCs into ExE cells by transcription factors. The ExE differentiation process is divided into at least two stages. In the first stage, ESCs differentiate into COUP-TF1-positive PrE cells, and in the second stage, they differentiate further into XEN, VE and PE cells. We have demonstrated that Sox17 plays a substantial role in the late-stage differentiation.

 

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© The Company of Biologists Ltd 2007