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First published online September 7, 2007
doi: 10.1242/10.1242/dev.006569

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The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells

Fabrice Lavial¹, Hervé Acloque^1,*, Federica Bertocchini², David J. MacLeod³, Sharon Boast², Elodie Bachelard¹, Guillaume Montillet¹, Sandrine Thenot¹, Helen M. Sang³, Claudio D. Stern², Jacques Samarut¹ and Bertrand Pain^4,5,

¹ Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, CNRS, INRA, Ecole Normale Supérieure de Lyon, France.
² Department of Anatomy and Developmental Biology, University College London, London, UK.
³ Roslin Institute, Roslin, Midlothian EH25 9PS, UK.
⁴ Inserm, U384, Faculté de Médecine, 28, place Henri Dunant, Clermont-Ferrand, F-63001, France.
⁵ Département de Physiologie Animale, INRA, France.

View larger version (122K): [in this window] [in a new window]   Fig. 1. The cPouV gene encodes a chicken PouV protein. (A) Alignment of D. rerio Pou2 (NP_571187), X. laevis Pou91 (AAA49999.1), M. musculus Oct4 (Pou5f1, NP_038661) and H. sapiens OCT4 (POU5F1, NP_002692) PouV proteins with the chicken PouV coding sequence (DQ867024) using NTi Clustal software (Invitrogen). (B,C) Similarity table analysis of the full-length proteins (B) or of their homeodomains (C) of Oct4 homologues was performed with sequences of 360 aa for B. taurus (Bt) Oct4 (NP_777005), of 360 aa for Sus scrofa (Ss) Oct4 (Q9TSV5), of 360 aa for H. sapiens (Hs) OCT4 (NP_002692), of 360 aa for P. troglodytes (Pt) Oct4 (Q7YR49), of 352 aa for M. musculus (Mm) Oct4 (NP_038661), of 189 aa for T. vulpecula (Tv) Oct4 (AAQ24229), of 472 aa for D. rerio (Dr) Pou2 (NP_571187), of 448 aa for X. laevis (Xl) Pou25 (AAA49996), of 445 aa for X. laevis Pou91 (AAA49999) and of 426 aa for X. laevis Pou60 (AAA49997). The 295 aa were used for the G. gallus (Gg) PouV protein (DQ867024). (D) Similarity table analysis of Nkx and Nanog families performed with sequences of 344 aa for G. gallus (Gg) Nkx2.1 (NP_989947), of 354 aa for M. musculus (Mm) Nkx2.4 (NP_075993), of 323 aa for G. gallus Nkx2.3 (CAA66257), of 362 aa for M. musculus Nkx2.3 (NP_032725), of 294 aa for G. gallus Nkx2.5 (NP_990495), of 299 aa for X. laevis (Xl) Nkx2.5 (AAA19861), of 324 aa for H. sapiens (Hs) NKX2.5 (NP_004378), of 318 aa for M. musculus Nkx2.5 (NP_032726), of 216 aa for M. musculus Nkx2.6 (NP_035050), of 305 aa for M. musculus Nanog (BAC76998), of 312 aa for R. rattus (Rr) Nanog (XP_575662), of 305 aa for H. sapiens NANOG (NP_079141), of 232 aa for H. sapiens (Hs-2) NANOG2 (AAS57555), of 273 aa for H. sapiens NKX2.2 (NP_002500) and of 273 aa for M. musculus Nkx2.2 (NP_035049). The 310 aa were used for the chicken Nanog protein (DQ 867025). Red text and yellow highlights indicate a complete aa conservation between tested species; blue text and blue highlights indicate partial conservation between tested species.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Kinetics of expression of pluripotency genes during differentiation of cESC. Proliferating cESC were induced to differentiate (A) by retinoic acid treatment at 10-7 M for 5 days after plating or (D,E) by embryoid body formation for 4 days. Five independent experiments provided similar results. (B,C) As in A except that cycloheximide (B) or actinomycin D (C) was added to the culture medium at 10 µg/ml at T=0; two independent experiments provided similar results. Expression of some of the genes analysed, as measured by real-time RT-PCR, was downregulated (D) or upregulated (E). A value of 1 was assigned to expression levels at T=0, i.e. at the start of the induction of differentiation.

View larger version (75K): [in this window] [in a new window]   Fig. 3. cPouV expression during chick embryo development. (A-J) Whole-mount in situ hybridisation to cPouV transcripts. Transcripts are detected in the area pellucida and area opaca of the epiblast in pre-streak embryos (A, stage XI; B, stage XIII), and in the hypoblast in a salt-and-pepper manner (B'). At stage XIV (C), the expression is very strong in the area pellucida of the epiblast, especially where the streak is forming (C'). Transcripts are expressed in the ingressing mesoderm at stages 2-3 (D,D'). As the primitive streak elongates and the embryo grows, expression is still detected in the ectoderm and mesoderm (E, stage 3+; F,F', stage 4+; G, stage 5). At stage 7 (H) and 8 (I), cPouV mRNA is detected in the forming neural tube and in the underlying mesoderm, but is absent from the endoderm (data not shown). At stage 9 (J), cPouV mRNA is expressed in neural tissue (nt) and presumptive migrating germ cells (gc). B',C',D',F' are transverse sections of the embryos in B,C,D,F, respectively. ao, area opaca; ep, epiblast; gc, germ cells; hy, hypoblast; m, mesoderm; np, neural plate; nt, neural tube; ps, primitive streak.

View larger version (147K): [in this window] [in a new window]   Fig. 4. cNanog expression during chick embryo development. (A-J) Whole-mount in situ hybridisation to cNanog transcripts. Nanog transcripts are localised in the epiblast of the area pellucida and area opaca of the pre-streak embryo (A, stage XI; B, stage XII), but not in the hypoblast (B'). From stage XIV, cNanog mRNA disappears from the posterior area pellucida (C,C') and from the growing primitive streak (D, stage 3; E,E', stage 3+; F, stage 4+). cNanog transcripts are downregulated in the epiblast from stage 4+ (F), and are confined anteriorly in a crescent region in the epiblast (G, stage 5+). At stage 6 (H), expression is restricted to the neural plate and the neural tube (I, stage 7; J, stage 8). cNanog is also expressed in scattered cells in the germinal crescent from stage 4 (arrowhead in I and H'). B' is a longitudinal section of the embryo in B, anterior at the right; C',E',H' are transverse sections of embryos in C,E,H, respectively. ep, epiblast; gd, gonad; ms, mesonephros; np, neural plate; nt, neural tube; ps, primitive streak.

View larger version (97K): [in this window] [in a new window]   Fig. 5. cPouV and cNanog are expressed in germ cells during later embryonic development. (A-H) At stage 33, cPouV mRNA is detected in the developing gonad, which is attached to the mesonephros (A,B), in the germ cells (D), as detected by co-localisation (F) of cPouV (D) both with Cvh (chicken Vasa, E) expression and with SSEA-1-positive cells (G) revealed on adjacent sections counterstained by Hoechst and cPouV probe (H, arrows). (B) Section of gonad shown in A. (C) Bright field of the stage 33 gonad used for in situ hybridisation (D,E,F). (I-M) At stage 33, cNanog is highly expressed in gonads and in mesonophros tubules (I) and gonad (I,J), especially in germ cells (K), as revealed by SSEA-1 staining (L) on the same cells that express cNanog in adjacent sections (M, arrow) counterstained by Hoechst (L). (K) Section of dissected gonad from the urogenital tract (J). ms, mesonephros; gd, gonads. Scale bars: 15 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 6. Overexpression of cPouV in cESC and mESC. (A) Following transfection of the different PouV coding sequences, overexpression of cPouV in cESC revealed loss of endogenous cNanog expression and an increase in Gata4, Gata6 and Cdx2 expression. (B) A similar profile was observed for XlPou91, but only a slight increase in Cdx2 when mOct4 is transfected in cESC. (C,D) Upregulation of Gata4, Gata6 and Cdx2 is strong (C) when XlPou91 is overexpressed in mESC with an induction of other markers (Hnf1, brachyury, Sox17 and laminin B1), in contrast to a moderate induction when cPouV is overexpressed in mESC (D). A value of 1 was given to the gene expression level obtained in clones transfected with the empty vector (empty). Two independent experiments provided similar results.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Oct4-deficient ZHBTc4 mESC are only partially rescued by cPouV expression. ZHBTc4 cells transfected with mOct4, XlPou91, cPouV or pou2 expression vectors were treated with doxycyclin after selection of stable clones. AP-positive clones were obtained with mOct4 (A), XlPou91 (B) and cPouV (C), but no clones were isolated with empty control vector (empty) or pou2. A rescue index (RI, the ratio between the number of clones in the presence versus the absence of doxycyclin) of 1 is given in the presence of mOct4 (D). This RI is the result of two independent experiments with a total number of clones of 114/0, 82/19, 123/95, 74/8 and 82/0, respectively, in the absence/presence of doxycylin for the empty vector, the mOct4, XlPou91, cPouV and pou2 expression vectors. (E) Expression of pluripotency-associated genes in ZHBTc4 complemented clones was analysed by real-time RT-PCR. A value of 1 was given to the level detected in the mOct4-complemented clones. Nanog, Utf1, Zfp42 (Rex1) and Tert expression was lower in clones complemented by cPouV than in those complemented with XlPou91. (F) ZHBTc4 cells were co-transfected in the presence of luciferase reporter gene driven either by the PE promoter or the Oct4 consensus sequence (ATGCAAAT). A value of 1 was given to the empty vector. Each result is the average of four wells per condition, and two independent experiments provided similar results. cPouV expression activated both promoters.

View larger version (48K): [in this window] [in a new window]   Fig. 8. Overexpression of cNanog induces growth factor-independent proliferation in both mESC and cESC. mESC or cESC were transfected with either mNanog, cNanog or empty expression vector. For mESC, LIF was removed, clones scored (A), stained for alkaline phosphatase activity and observed (B-G). Similar results were obtained in two independent experiments. Scale bars: 400 µm. (H) Gene expression analysis revealed expression of mOct4 as well as of Sox2, Utf1, Rex1 and Tert, but with the notable exception of Fgf4. A value of 1 was given to the level of expression in the clones obtained without LIF in the presence of mNanog. (I) For cESC, growth factors and cytokines (bFgf, Scf, Igf1, Il6, Il6R) (-F) were removed, serum concentration reduced as indicated (10% to 1%) and clones scored. (J,K) Gene expression analysis revealed maintenance of cPouV expression in the presence of 10% serum without factors (J), but a complete loss of expression when serum was reduced to 1% without factors (K). Under these conditions, the expression of cNanog is upregulated and the cells continue to proliferate.

View larger version (69K): [in this window] [in a new window]   Fig. 9. Inhibition of cPouV and cNanog expression stops proliferation and induces cESC to differentiate. cESC were transfected with vectors allowing conditional expression of shRNA-2 and shRNA-1 against cPouV and Nanog, respectively, and of Cre-ERT2 recombinase, the activity of which was induced by 4-hydroxytamoxifen. Similar results were obtained with shRNA-3 against cNanog. The morphology of the cells targeted for cPouV (C,D) and cNanog (E,F) was strongly altered, compared with control cells (A,B) and with targeted cells for cOct6 (data not shown), in 60% of the clones (G). In these differentiated cells, the percentage of SSEA-1-positive cells was drastically reduced (H). Proliferation was also rapidly reduced as assessed by XTT proliferation on 12 independent clones followed for 4 days after tamoxifen addition (T=0 hours) (I). Gene expression analysis (J) revealed a strong decrease in expression of target genes cPouV and cNanog in contrast to a strong upregulation of Gata4, Gata6 and Cdx2 in cells expressing shRNA against cPouV, and the upregulation of Gata4 and Gata6 for those cells expressing shRNA against cNanog. In the latter case, no Cdx2 expression could be detected. A shRNA-3 against another Pou family member, Oct6, does not alter cPouV and cNanog expression by comparison with the control vector (empty). Oct6 expression cannot be detected in the cells targeted by shRNA against cNanog (J). Three independent experiments provided similar results.

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