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Antagonism between Smad1 and Smad2 signaling determines the site of distal visceral endoderm formation in the mouse embryo.

Yamamoto M, Beppu H, Takaoka K, Meno C, Li E, Miyazono K, Hamada H - J. Cell Biol. (2009)

Bottom Line: A Smad2-activating factor such as Activin also contributes to DVE formation by generating a region of VE positive for the Smad2 signal and negative for Smad1 signal.DVE is thus formed at the distal end of the embryo, the only region of VE negative for the Smad1 signal and positive for Smad2 signal.An inverse relation between the level of phosphorylated Smad1 and that of phosphorylated Smad2 in VE suggests an involvement of antagonism between Smad1- and Smad2-mediated signaling.

View Article: PubMed Central - PubMed

Affiliation: Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan. myamamoto@fbs.osaka-u.ac.jp

ABSTRACT
The anterior-posterior axis of the mouse embryo is established by formation of distal visceral endoderm (DVE) and its subsequent migration. The precise mechanism of DVE formation has remained unknown, however. Here we show that bone morphogenetic protein (BMP) signaling plays dual roles in DVE formation. BMP signaling is required at an early stage for differentiation of the primitive endoderm into the embryonic visceral endoderm (VE), whereas it inhibits DVE formation, restricting it to the distal region, at a later stage. A Smad2-activating factor such as Activin also contributes to DVE formation by generating a region of VE positive for the Smad2 signal and negative for Smad1 signal. DVE is thus formed at the distal end of the embryo, the only region of VE negative for the Smad1 signal and positive for Smad2 signal. An inverse relation between the level of phosphorylated Smad1 and that of phosphorylated Smad2 in VE suggests an involvement of antagonism between Smad1- and Smad2-mediated signaling.

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Identification of BMP as the inhibitory signal from ExE that restricts DVE formation. The DVE region was monitored by detection of the expression of a Hex-Venus transgene (B, D, Q, R, T, U, W, and X, green) at the indicated times after removal of ExE (B and D) or after ExE attachment (Q, R, T, U, W, and X) at E5.5. Embryo explants stripped of ExE (A and C) or normal embryos (K–N) at E5.5 were subjected to immunohistofluorescence staining with antibodies to p-Smad1 (pS1; green) or to p-Smad2 (pS2; green), as indicated. Merged images with nuclear staining (Nuc; red) are also shown. Green color in merged images indicates increased level of p-Smad1 or p-Smad2 staining. The explants or embryos were cultured with BSA (A, B, K, and M), BMP4 (C and D), or Noggin (Nog; L and N) for 6 h or the indicated times. Expression of Lefty1 (L1), Cerl, and Lim1 was determined in E5.2 embryos cultured for 8 h with either BSA (E, G, and I) or Noggin (F, H, and J). The experimental strategy for the explant experiments involving removal and attachment of ExE (P–X) is shown in O. Chimeric explants were composed of an embryonic portion harboring the Hex-Venus transgene and an extraembryonic portion treated with BSA or Noggin, as indicated. Bright-field images alone are shown in P, S, and V. Fluorescence images superimposed on bright-field images (B, D, Q, R, T, U, W, and X) are also shown. Bars, 50 µm.
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fig6: Identification of BMP as the inhibitory signal from ExE that restricts DVE formation. The DVE region was monitored by detection of the expression of a Hex-Venus transgene (B, D, Q, R, T, U, W, and X, green) at the indicated times after removal of ExE (B and D) or after ExE attachment (Q, R, T, U, W, and X) at E5.5. Embryo explants stripped of ExE (A and C) or normal embryos (K–N) at E5.5 were subjected to immunohistofluorescence staining with antibodies to p-Smad1 (pS1; green) or to p-Smad2 (pS2; green), as indicated. Merged images with nuclear staining (Nuc; red) are also shown. Green color in merged images indicates increased level of p-Smad1 or p-Smad2 staining. The explants or embryos were cultured with BSA (A, B, K, and M), BMP4 (C and D), or Noggin (Nog; L and N) for 6 h or the indicated times. Expression of Lefty1 (L1), Cerl, and Lim1 was determined in E5.2 embryos cultured for 8 h with either BSA (E, G, and I) or Noggin (F, H, and J). The experimental strategy for the explant experiments involving removal and attachment of ExE (P–X) is shown in O. Chimeric explants were composed of an embryonic portion harboring the Hex-Venus transgene and an extraembryonic portion treated with BSA or Noggin, as indicated. Bright-field images alone are shown in P, S, and V. Fluorescence images superimposed on bright-field images (B, D, Q, R, T, U, W, and X) are also shown. Bars, 50 µm.

Mentions: To examine whether the ExE-derived inhibitory signal is indeed the BMP signal, we examined the response of DVE to BMP or a BMP inhibitor. When wild-type embryos were cultured from E5.2 to E5.5 in the presence of BMP4, expression of Hex, Lefty1, and Cerl was lost (Fig. S5, A–F, available at http://www.jcb.org/cgi/content/full/jcb.200808044/DC1). Whereas staining for p-Smad1 was lost in VE in response to removal of ExE at E5.5 (Fig. 6 A), p-Smad1 staining was maintained in VE if the explants lacking the ExE were cultured with BMP4 (Fig. 6 C). Moreover, expansion of the DVE region in such explants was inhibited by BMP4 (Fig. 6, B and D; and Fig. S5, T–W). In contrast, the region positive for DVE markers expanded and the level of expression of DVE markers increased when whole embryos at E5.2 were cultured with Noggin (Fig. 6, E–J), which induced down-regulation of p-Smad1 (Fig. 6, K and L).


Antagonism between Smad1 and Smad2 signaling determines the site of distal visceral endoderm formation in the mouse embryo.

Yamamoto M, Beppu H, Takaoka K, Meno C, Li E, Miyazono K, Hamada H - J. Cell Biol. (2009)

Identification of BMP as the inhibitory signal from ExE that restricts DVE formation. The DVE region was monitored by detection of the expression of a Hex-Venus transgene (B, D, Q, R, T, U, W, and X, green) at the indicated times after removal of ExE (B and D) or after ExE attachment (Q, R, T, U, W, and X) at E5.5. Embryo explants stripped of ExE (A and C) or normal embryos (K–N) at E5.5 were subjected to immunohistofluorescence staining with antibodies to p-Smad1 (pS1; green) or to p-Smad2 (pS2; green), as indicated. Merged images with nuclear staining (Nuc; red) are also shown. Green color in merged images indicates increased level of p-Smad1 or p-Smad2 staining. The explants or embryos were cultured with BSA (A, B, K, and M), BMP4 (C and D), or Noggin (Nog; L and N) for 6 h or the indicated times. Expression of Lefty1 (L1), Cerl, and Lim1 was determined in E5.2 embryos cultured for 8 h with either BSA (E, G, and I) or Noggin (F, H, and J). The experimental strategy for the explant experiments involving removal and attachment of ExE (P–X) is shown in O. Chimeric explants were composed of an embryonic portion harboring the Hex-Venus transgene and an extraembryonic portion treated with BSA or Noggin, as indicated. Bright-field images alone are shown in P, S, and V. Fluorescence images superimposed on bright-field images (B, D, Q, R, T, U, W, and X) are also shown. Bars, 50 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654303&req=5

fig6: Identification of BMP as the inhibitory signal from ExE that restricts DVE formation. The DVE region was monitored by detection of the expression of a Hex-Venus transgene (B, D, Q, R, T, U, W, and X, green) at the indicated times after removal of ExE (B and D) or after ExE attachment (Q, R, T, U, W, and X) at E5.5. Embryo explants stripped of ExE (A and C) or normal embryos (K–N) at E5.5 were subjected to immunohistofluorescence staining with antibodies to p-Smad1 (pS1; green) or to p-Smad2 (pS2; green), as indicated. Merged images with nuclear staining (Nuc; red) are also shown. Green color in merged images indicates increased level of p-Smad1 or p-Smad2 staining. The explants or embryos were cultured with BSA (A, B, K, and M), BMP4 (C and D), or Noggin (Nog; L and N) for 6 h or the indicated times. Expression of Lefty1 (L1), Cerl, and Lim1 was determined in E5.2 embryos cultured for 8 h with either BSA (E, G, and I) or Noggin (F, H, and J). The experimental strategy for the explant experiments involving removal and attachment of ExE (P–X) is shown in O. Chimeric explants were composed of an embryonic portion harboring the Hex-Venus transgene and an extraembryonic portion treated with BSA or Noggin, as indicated. Bright-field images alone are shown in P, S, and V. Fluorescence images superimposed on bright-field images (B, D, Q, R, T, U, W, and X) are also shown. Bars, 50 µm.
Mentions: To examine whether the ExE-derived inhibitory signal is indeed the BMP signal, we examined the response of DVE to BMP or a BMP inhibitor. When wild-type embryos were cultured from E5.2 to E5.5 in the presence of BMP4, expression of Hex, Lefty1, and Cerl was lost (Fig. S5, A–F, available at http://www.jcb.org/cgi/content/full/jcb.200808044/DC1). Whereas staining for p-Smad1 was lost in VE in response to removal of ExE at E5.5 (Fig. 6 A), p-Smad1 staining was maintained in VE if the explants lacking the ExE were cultured with BMP4 (Fig. 6 C). Moreover, expansion of the DVE region in such explants was inhibited by BMP4 (Fig. 6, B and D; and Fig. S5, T–W). In contrast, the region positive for DVE markers expanded and the level of expression of DVE markers increased when whole embryos at E5.2 were cultured with Noggin (Fig. 6, E–J), which induced down-regulation of p-Smad1 (Fig. 6, K and L).

Bottom Line: A Smad2-activating factor such as Activin also contributes to DVE formation by generating a region of VE positive for the Smad2 signal and negative for Smad1 signal.DVE is thus formed at the distal end of the embryo, the only region of VE negative for the Smad1 signal and positive for Smad2 signal.An inverse relation between the level of phosphorylated Smad1 and that of phosphorylated Smad2 in VE suggests an involvement of antagonism between Smad1- and Smad2-mediated signaling.

View Article: PubMed Central - PubMed

Affiliation: Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan. myamamoto@fbs.osaka-u.ac.jp

ABSTRACT
The anterior-posterior axis of the mouse embryo is established by formation of distal visceral endoderm (DVE) and its subsequent migration. The precise mechanism of DVE formation has remained unknown, however. Here we show that bone morphogenetic protein (BMP) signaling plays dual roles in DVE formation. BMP signaling is required at an early stage for differentiation of the primitive endoderm into the embryonic visceral endoderm (VE), whereas it inhibits DVE formation, restricting it to the distal region, at a later stage. A Smad2-activating factor such as Activin also contributes to DVE formation by generating a region of VE positive for the Smad2 signal and negative for Smad1 signal. DVE is thus formed at the distal end of the embryo, the only region of VE negative for the Smad1 signal and positive for Smad2 signal. An inverse relation between the level of phosphorylated Smad1 and that of phosphorylated Smad2 in VE suggests an involvement of antagonism between Smad1- and Smad2-mediated signaling.

Show MeSH