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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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DVE formation requires BMP signaling in the extraembryonic region. Expression of Lefty1 (A and A'), Cerl (B and B'), Dkk1 (C and C'), Lim1 (D and D'), Hex (E, E', I, and I'), Hnf4 (J and J'), Pem (K and K'), Bmp2 (L and L'), and Fgf8 (M, M', and M”) was examined by in situ hybridization in wild-type (Bmpr2+/+) and Bmpr2−/− mouse embryos at E5.5 or the indicated stages. The DVE was absent (A'–E') in the mutant embryos. The primitive endoderm (I') and extraembryonic VE (J' and K') were formed, whereas embryonic VE was absent (L' and M') or impaired (M”). Arrowheads in J' indicate the junction between the extraembryonic and embryonic regions. Green ES FM260 cell (Bmpr2+/+)←→Bmpr2+/+ tetraploid (F–H) and green ES FM260 cell←→Bmpr2−/− tetraploid (F'–H') chimeric embryos were recovered at E6.5 and examined for EGFP fluorescence (F and F') or for expression of Hex (G and G') and Lefty1 (H and H'). The expression of Hex and Lefty1 was absent in the green ES FM260 cell←→Bmpr2−/− tetraploid chimeric embryos. (N) The level of p-Smad1 staining and the level of expression of DVE markers are compared between Bmpr2−/− and Bmpr2−/−,Actr2b+/− embryos. Dark blue, light blue, orange, and red bars indicate normal expression, expression at a moderately reduced level, expression at a severely reduced level, and no expression, respectively. The numbers of embryos showing each expression pattern are indicated. Bars, 50 µm.
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fig1: DVE formation requires BMP signaling in the extraembryonic region. Expression of Lefty1 (A and A'), Cerl (B and B'), Dkk1 (C and C'), Lim1 (D and D'), Hex (E, E', I, and I'), Hnf4 (J and J'), Pem (K and K'), Bmp2 (L and L'), and Fgf8 (M, M', and M”) was examined by in situ hybridization in wild-type (Bmpr2+/+) and Bmpr2−/− mouse embryos at E5.5 or the indicated stages. The DVE was absent (A'–E') in the mutant embryos. The primitive endoderm (I') and extraembryonic VE (J' and K') were formed, whereas embryonic VE was absent (L' and M') or impaired (M”). Arrowheads in J' indicate the junction between the extraembryonic and embryonic regions. Green ES FM260 cell (Bmpr2+/+)←→Bmpr2+/+ tetraploid (F–H) and green ES FM260 cell←→Bmpr2−/− tetraploid (F'–H') chimeric embryos were recovered at E6.5 and examined for EGFP fluorescence (F and F') or for expression of Hex (G and G') and Lefty1 (H and H'). The expression of Hex and Lefty1 was absent in the green ES FM260 cell←→Bmpr2−/− tetraploid chimeric embryos. (N) The level of p-Smad1 staining and the level of expression of DVE markers are compared between Bmpr2−/− and Bmpr2−/−,Actr2b+/− embryos. Dark blue, light blue, orange, and red bars indicate normal expression, expression at a moderately reduced level, expression at a severely reduced level, and no expression, respectively. The numbers of embryos showing each expression pattern are indicated. Bars, 50 µm.

Mentions: Formation of the primitive streak is impaired in Bmpr2−/− embryos (Beppu et al., 2000). To determine whether formation of the A-P axis occurs normally in these mutant embryos, we first examined the expression of AVE or DVE marker genes at E6.5 and E5.5, respectively. In wild-type embryos at E5.5, five DVE marker genes, Lefty1, Cerl, Dkk1, Lim1, and Hex, are expressed in VE at the distal tip (Fig. 1, A–E). Expression of Hex, Hesx1, and Cerl is absent at E5.2 but is apparent at E5.5 (Fig. S1, A–C and E–G, available at http://www.jcb.org/cgi/content/full/jcb.200808044/DC1), whereas Lefty1 expression is maintained between E4.0 and E5.5 (Takaoka et al., 2006; Fig. S1, D and H), indicating that cells positive for a full range of DVE markers are formed between E5.2 and E5.5. In Bmpr2−/− embryos, however, expression of AVE marker genes at E6.5 was absent or reduced compared with that in wild-type embryos (Fig. S2, A–D, A'–D', and A''–D''). Dkk1 expression was lost (Fig. S2 C') or remained relatively normal (Fig. S2 C''). At E5.5, expression of Lefty1, Cerl, Dkk1, and Lim1 was absent (4/7, 3/7, 3/7, and 3/6 embryos, respectively) or markedly reduced (3/7, 4/7, 4/7, and 3/6 embryos, respectively), and that of Hex was also lost (3/3 embryos; Fig. 1, A'–E' and N; and Fig. S2, I and I').


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)

DVE formation requires BMP signaling in the extraembryonic region. Expression of Lefty1 (A and A'), Cerl (B and B'), Dkk1 (C and C'), Lim1 (D and D'), Hex (E, E', I, and I'), Hnf4 (J and J'), Pem (K and K'), Bmp2 (L and L'), and Fgf8 (M, M', and M”) was examined by in situ hybridization in wild-type (Bmpr2+/+) and Bmpr2−/− mouse embryos at E5.5 or the indicated stages. The DVE was absent (A'–E') in the mutant embryos. The primitive endoderm (I') and extraembryonic VE (J' and K') were formed, whereas embryonic VE was absent (L' and M') or impaired (M”). Arrowheads in J' indicate the junction between the extraembryonic and embryonic regions. Green ES FM260 cell (Bmpr2+/+)←→Bmpr2+/+ tetraploid (F–H) and green ES FM260 cell←→Bmpr2−/− tetraploid (F'–H') chimeric embryos were recovered at E6.5 and examined for EGFP fluorescence (F and F') or for expression of Hex (G and G') and Lefty1 (H and H'). The expression of Hex and Lefty1 was absent in the green ES FM260 cell←→Bmpr2−/− tetraploid chimeric embryos. (N) The level of p-Smad1 staining and the level of expression of DVE markers are compared between Bmpr2−/− and Bmpr2−/−,Actr2b+/− embryos. Dark blue, light blue, orange, and red bars indicate normal expression, expression at a moderately reduced level, expression at a severely reduced level, and no expression, respectively. The numbers of embryos showing each expression pattern are indicated. Bars, 50 µm.
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fig1: DVE formation requires BMP signaling in the extraembryonic region. Expression of Lefty1 (A and A'), Cerl (B and B'), Dkk1 (C and C'), Lim1 (D and D'), Hex (E, E', I, and I'), Hnf4 (J and J'), Pem (K and K'), Bmp2 (L and L'), and Fgf8 (M, M', and M”) was examined by in situ hybridization in wild-type (Bmpr2+/+) and Bmpr2−/− mouse embryos at E5.5 or the indicated stages. The DVE was absent (A'–E') in the mutant embryos. The primitive endoderm (I') and extraembryonic VE (J' and K') were formed, whereas embryonic VE was absent (L' and M') or impaired (M”). Arrowheads in J' indicate the junction between the extraembryonic and embryonic regions. Green ES FM260 cell (Bmpr2+/+)←→Bmpr2+/+ tetraploid (F–H) and green ES FM260 cell←→Bmpr2−/− tetraploid (F'–H') chimeric embryos were recovered at E6.5 and examined for EGFP fluorescence (F and F') or for expression of Hex (G and G') and Lefty1 (H and H'). The expression of Hex and Lefty1 was absent in the green ES FM260 cell←→Bmpr2−/− tetraploid chimeric embryos. (N) The level of p-Smad1 staining and the level of expression of DVE markers are compared between Bmpr2−/− and Bmpr2−/−,Actr2b+/− embryos. Dark blue, light blue, orange, and red bars indicate normal expression, expression at a moderately reduced level, expression at a severely reduced level, and no expression, respectively. The numbers of embryos showing each expression pattern are indicated. Bars, 50 µm.
Mentions: Formation of the primitive streak is impaired in Bmpr2−/− embryos (Beppu et al., 2000). To determine whether formation of the A-P axis occurs normally in these mutant embryos, we first examined the expression of AVE or DVE marker genes at E6.5 and E5.5, respectively. In wild-type embryos at E5.5, five DVE marker genes, Lefty1, Cerl, Dkk1, Lim1, and Hex, are expressed in VE at the distal tip (Fig. 1, A–E). Expression of Hex, Hesx1, and Cerl is absent at E5.2 but is apparent at E5.5 (Fig. S1, A–C and E–G, available at http://www.jcb.org/cgi/content/full/jcb.200808044/DC1), whereas Lefty1 expression is maintained between E4.0 and E5.5 (Takaoka et al., 2006; Fig. S1, D and H), indicating that cells positive for a full range of DVE markers are formed between E5.2 and E5.5. In Bmpr2−/− embryos, however, expression of AVE marker genes at E6.5 was absent or reduced compared with that in wild-type embryos (Fig. S2, A–D, A'–D', and A''–D''). Dkk1 expression was lost (Fig. S2 C') or remained relatively normal (Fig. S2 C''). At E5.5, expression of Lefty1, Cerl, Dkk1, and Lim1 was absent (4/7, 3/7, 3/7, and 3/6 embryos, respectively) or markedly reduced (3/7, 4/7, 4/7, and 3/6 embryos, respectively), and that of Hex was also lost (3/3 embryos; Fig. 1, A'–E' and N; and Fig. S2, I and I').

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
Related in: MedlinePlus