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First published online 19 November 2003
doi: 10.1242/dev.00870

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Cadherin-mediated cell interaction regulates germ cell determination in mice

¹ Department of Molecular Embryology, Research Institute, Osaka Medical Center for Maternal and Child Health, 840, Murodo-cho, Izumi, Osaka, 594-1101, Japan
² Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
³ CREST, JST, Saitama, Japan
⁴ Department of Molecular Cell Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan

View larger version (103K): [in a new window]   Fig. 1. Localization of PGC precursor cells in early streak embryos. Explants of proximal epiblast at E6.5 (A) and E6.75 (D) cultured with recombinant human BMP4 for 72 and 66 hours, respectively. BMP4 stimulates PGC formation from E6.5 proximal epiblasts (unpublished results). ALP- and PGC7/stella-positive PGCs developed from E6.5 explants (B,C, arrowheads), but no such cells emerged from E6.75 explants (D). The same group of cells is shown in B and C. E6.75 extra-embryonic mesoderm generated ALP-positive PGCs (E). Scale bar: 160 µm (A,D,E) and 100 µm (B,C).

View larger version (82K): [in a new window]   Fig. 2. Expression of Oct4 (GOF18-deltaPE)-GFP transgenes in gastrulating embryos. Fluorescence images of embryos at (A) E6.5 (early streak stage), (B) E6.75 (mid-streak stage), (C) E7.25 (early allantoic bud stage). (D,E,F) Extra-embryonic mesoderm of Oct4-GFP embryos at mid-allantoic bud stage showing expression of Oct4-GFP (D arrowheads) and stained with anti-PGC7 antibody (E). (F) Merged image. The cells that express intense Oct4-GFP (D, filled arrowheads) in base of allantoic bud also stained with anti-PGC7 antibody (E, open arrowheads). Scale bar: 200 µm (A-C); 25 µm (D-F).

View larger version (69K): [in a new window]   Fig. 3. PGCs from Oct4-positive cell cluster in isolated extra-embryonic mesoderm at E6.75. (A-G) Time-lapse analysis of Oct4-GFP extra-embryonic mesoderm. (A-E,G) Images of cells expressing Oct4-GFP in extra-embryonic mesoderm on an STO feeder layer after (A) 0, (B) 12, (C) 15, (D) 30, (E) 36 and (G) 40 hours in culture. (F) ALP expression of explant in G. Arrowheads indicate ALP- and GFP-positive migrating PGCs. (H,I,J) Co-expression of PGC markers, (H) ALP, (I) PGC7 and (J) Oct4-GFP in PGCs (arrowheads) that developed from cultured E6.75 extra-embryonic mesoderm. Scale bar: 200 µm (A-G) and 100 µm (H-J).

View larger version (42K): [in a new window]   Fig. 7. Specific expression of E-cadherin within the cluster of PGC precursors. Explants of Oct4-GFP extra-embryonic mesoderm at E6.75 were cultured for 9-10 hours, and stained with anti-PGC7 antibody (B) or with anti-E-cadherin antibody, ECCD-2 (E). Oct4-GFP expression (A,D). (C,F) Merged images with TOTO-3 stained nuclei. Cluster of GFP-expressing PGC precursors that are PGC7/stella negative exclusively maintained E-cadherin expression. Scale bar: 50 µm.

View larger version (52K): [in a new window]   Fig. 4. PGCs from E7.25 extra-embryonic mesoderm. Intact extra-embryonic mesoderm fragment (A) and cells dissociated from a fragment (B,C) were cultured for 48 hours, and then stained for ALP activity (A,B) or for PGC7 (C). ALP-positive cells developed from the intact fragment (A) and from dissociated cells (B, arrowheads). ALP-positive cells in B also stained with the anti-PGC7 antibody (C, arrowheads). Scale bars, 160 µm (A) and 100 µm (B,C) µm.

View larger version (97K): [in a new window]   Fig. 5. E-cadherin expression in clustered PGC precursors at base of nascent allantois. Lateral view of embryos at (A-D) mid-streak (E6.75), (E-H) mid- to late streak (E7.0), and (I-L) early allantoic bud (E7.25) stage with anterior to left. (A-D) Oct4-GFP (A) and E-cadherin (B) expression was slightly and obviously lower in the distal portion of extra-embryonic mesoderm (dotted line). Arrows indicate boundary of epiblast and extra-embryonic mesoderm; exee, extra-embryonic ectoderm; exem, extra-embryonic mesoderm; epi, epiblast; ve, visceral endoderm. (E-H) Cells expressing intense GFP (E, solid line) form a cluster at the apical tip of the E-cadherin-expressing region (F) of extra-embryonic mesoderm. (I-L) Committed PGCs expressing PGC7 (I) dispersed in the most distal region in E-cadherin-expressing extra-embryonic mesoderm (J). (C,G,K) TOTO-3 staining of nuclei and (D,H,L) merged images. Scale bar: 50 µm (A-D,I-L) and 25 µm (E-H).

View larger version (114K): [in a new window]   Fig. 6. Antibody blocking of E-cadherin impairs PGC formation in culture. Explants of Oct4-GFP extra-embryonic mesoderm at E6.75 were cultured for 15 hours with no antibody (A-D,M-P), with ECCD-1 that blocks E-cadherin (E-H), or with ECCD-2, a control antibody with no blocking function (I-L). Cultured explants were stained with anti-PGC7 antibody (B,F,J) or with anti-E-cadherin, ECCD-2 (N). (A,E,I,M) Oct4-GFP expression; (C,G,K,O) merged images; (D,H,L,P) phase contrast images. With ECCD-1, scattered Oct4-GFP-positive cells are evident, but they are PGC7 negative (E-G). Oct4-GFP positive clump of cells express E-cadherin, while the remaining explant is E-cadherin-negative in control culture (M-P). Scale bar: 50 µm (A-D,M-P); 80 µm (E,L).

View larger version (50K): [in a new window]   Fig. 8. Extra-embryonic mesoderm develops normally in cultured explants with E-cadherin-blocking antibody. Explants of extra-embryonic mesoderm were cultured for 15 hours with ECCD-1 (A-D), or with ECCD-2 (E-H). Cultured explants were stained with anti-PGC7 (A,E) and anti-Flk1 antibodies to show differentiation of extra-embryonic mesoderm (B, F). (C,G) Merged and (D,H) phase contrast images. Scale bar: 100 µm.

View larger version (134K): [in a new window]   Fig. 9. Inhibition of migrating PGC emergence by E-cadherin-blocking antibody in prolonged culture. Extra-embryonic mesoderm explants at E6.75 (A,B) and E7.25 (C,D) were cultured with ECCD-1 (A,C) or ECCD-2 (B,D) for 48-60 hours. Emergence of PGCs was detected by ALP staining. Scale bar: 200 µm.

View larger version (22K): [in a new window]   Fig. 10. Possible mechanism of PGC determination by E-cadherin-mediated cell interaction. PGC/allantois common precursors locate within the proximal region of extra-embryonic mesoderm. Cells losing E-cadherin expression move to the distal region of the extra-embryonic mesoderm and differentiate into allantois. In contrast, some cells continuously expressing E-cadherin form clusters and E-cadherin-mediated cell signaling directs them to germ cell fate. exee, extra-embryonic ectoderm.

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