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First published online March 7, 2005
doi: 10.1242/10.1242/dev.01715

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Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts

Tilo Kunath^1,2,*, Danielle Arnaud³, Gary D. Uy⁴, Ikuhiro Okamoto⁵, Corinne Chureau³, Yojiro Yamanaka¹, Edith Heard⁵, Richard L. Gardner⁴, Philip Avner^3, and Janet Rossant^1,2,

¹ Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G 1X5, Canada
² Department of Molecular and Medical Genetics, University of Toronto, Toronto M5S 1A8, Canada
³ Unité de Génétique Moléculaire Murine, URA 2578 CNRS, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, France
⁴ Mammalian Development Laboratory, Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK
⁵ CNRS UMR218, Curie Institute, 26 rue d'Ulm, Paris 75005, France

View larger version (97K): [in a new window]   Fig. 1. The extra-embryonic endoderm lineage and XEN cell cultures. (A) Schematic of an E4.5 blastocyst and an E5.5 embryo, illustrating the primitive, visceral and parietal endoderm (green). The trophoblast and epiblast lineages are represented in yellow and red, respectively. The proximodistal axis is indicated. ICM, inner cell mass. (B) Phase-contrast micrograph of XEN cells cultured at low density illustrating the refractile, rounded cell type and the epithelioid cell type. (C) XEN cells grown to near confluency showing the presence of individual cells and an epithelial sheet of cells. (D) XEN cells participating in a lattice-type structure. (E) XEN cells plated in the absence of gelatin resulted in differentiation of some cells into large vacuolated cells in 6 days. Scale bar: 10 µm for B-D; 40 µm for E.

View larger version (149K): [in a new window]   Fig. 2. Scanning electron microscopy of XEN cells and a pre-gastrula embryo. (A) Rounded XEN cell with numerous microvilli. (B) Epithelial-like XEN cell with numerous microvilli and lamellepodia at opposite ends. (C) Isolated XEN cell with a short pseudopodium. (D) Colony of XEN cells where one cell is extending several pseudopodia. (E) Isolated XEN cell with a curving pseudopodium viewed at a 60° angle. This XEN cell is on a pedestal structure and the pseudopodium is originating from below the cell. (F) E5.5 embryo with Reichert's membrane removed (inset) and a higher magnification of the anterior visceral endoderm region (broken line). Scale bars: 5 µm for A,E; 10 µm for B,C,F; 20 µm for D.

View larger version (82K): [in a new window]   Fig. 3. RT-PCR expression analysis of XEN cells. cDNA was prepared from ES cells, embryoid bodies (EB) at day 5 (D5) and day 7 (D7), and three different XEN cell cultures. XEN cells were either cultured on gelatin in maintenance conditions (XEN) or differentiated (Diff) by removal of EMFI-CM and gelatin for 4 or 8 days (D4, D8). Hnf4 and Foxa2 were consistently detected in all XEN cell cultures, while Gata4 and Sox7 decreased expression upon differentiation. By contrast, -fetoprotein (Afp) increased with differentiation. Oct4 was undetectable in XEN cells.

View larger version (95K): [in a new window]   Fig. 4. XEN cell chimeras. (A,B) E6.5 chimera with XEN cells contributions to the distal region of the conceptus in the parietal yolk sac: (A) Partial phase-contrast and UV fluorescence micrograph; (B) UV fluorescence micrograph with false-color added. (C,D) Two E7.5 chimeras with XEN cell contributions exclusively to the parietal yolk sac; phase-contrast (C) and UV fluorescence micrographs (D). (E) E8.5 XEN cell chimera in which the parietal yolk sac was dissected from the visceral yolk sac and embryo proper after X-gal staining. All the X-gal-positive cells were located in the parietal yolk sac and not in other tissues. (F) E7.5 XEN cell chimera after X-gal staining. A coherent XEN cell clone was observed in the extra-embryonic region of the visceral endoderm.

View larger version (14K): [in a new window]   Fig. 5. X-inactivation in ES, TS and XEN cells. Quantification of Xist transcripts by real-time RT-PCR in (A) ES cells and (B) XEN and TS cells normalized using the endogenous gene 18S. (C) Quantification of Nap1l2 transcripts normalized using the Rrm2 gene in XEN cells. In differentiated ES cells (A), random X-inactivation leads to equivalent levels of Xist expression from the 129 and Pgk alleles. By contrast, in female XEN cells (GHP7/3 and GHP7/9), Xist is expressed only from the paternal Pgk allele (B) and Nap1l2 only from the maternal 129 allele (C), indicating that the paternal X chromosome is preferentially inactivated in female XEN cells, just as in female TS cells (where Xist is expressed only from the paternal 129 allele; B). In control male XEN cells (GHP7/7), the single X chromosome derived from 129 is active, as shown by the absence of Xist (B) and presence of Nap1l2 expression (C).

View larger version (63K): [in a new window]   Fig. 6. Histone modifications and Xist accumulation in female XEN and TS cells. (A,B) Representative images show immunofluorescent detection of Eed, Ezh2 and histone modifications H3 di/tri-methyl K27 and H3 acetyl K9, combined with Xist RNA FISH. Immunodetections were performed using Alexa 568-conjugated secondary antibodies (red). Xist RNA was detected using a Spectrum Green-labeled FISH probe (green). XEN cells (A) show no Eed and Ezh2 enrichment, and weak H3 di/tri-meK27 enrichment on the Xist-coated X chromosome, but they do show depletion for H3 acK9. By contrast, undifferentiated TS cells (B) show enrichment for Eed, Ezh2 and H3 di/tri-meK27 on the Xist-coated X chromosome. Depletion for H3 acK9 was observed, as in XEN cells.