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1.
Fig. 7.

Fig. 7. From: Anteroposterior and dorsoventral patterning are coordinated by an identical patterning clock.

Model of coordinated patterning of DV and AP tissues. DV patterning by BMP signaling along the AP axis and temporally progressive AP patterning by Wnt, FGF and RA signaling are coordinated during gastrulation. FGF/MAPK inhibits P-Smad5 through phosphorylation of its linker region and thereby DV and AP tissues are temporally patterned progressively. As FGF/MAPK signaling within the margin moves vegetally (posteriorly) during epiboly progression, its inhibition of P-Smad1/5 becomes localized to progressively more posterior regions. Although Wnt and RA signaling remain within the margin during gastrulation, it is not known whether these signals act directly in temporally regulating BMP signaling in DV patterning. A, anterior; V, ventral; D, dorsal; P, posterior.

Megumi Hashiguchi, et al. Development. 2013 May 1;140(9):1970-1980.
2.
Fig. 6.

Fig. 6. From: Anteroposterior and dorsoventral patterning are coordinated by an identical patterning clock.

FGF/MAPK affects temporal patterning of DV tissues through the P-Smad5 linker region. (A-D) hoxb1b expression in Tg(hsp70:chd) embryos with HS at 65% epiboly. Lateral views, dorsal to right at 90-100% epiboly stage. Embryos were injected with smad5 MOs (B), smad5 MOs and hSmad1WT (hS1WT) mRNA (C) or smad5 MOs and hSmad1 MAPK mutant (hS1MM) mRNA (D). (E) The percentage of embryos exhibiting a fully dorsalized, weakly dorsalized, wild-type or ventralized phenotype in the various conditions. (F-I) otx2 and ntl expression in Tg(hsp70:chd) embryos with HS at 40% epiboly. Lateral views, dorsal to right at 80% epiboly. Embryos were injected with smad5 MOs (G), smad5 MOs and hS1WT mRNA (H) or smad5 MOs and hS1MM mRNA (I). (J) The percentage of embryos exhibiting the different dorsalization strength phenotypes in the various conditions. The panel borders in A-D and F-I match the phenotype colors in E and J.

Megumi Hashiguchi, et al. Development. 2013 May 1;140(9):1970-1980.
3.
Fig. 4.

Fig. 4. From: Anteroposterior and dorsoventral patterning are coordinated by an identical patterning clock.

Posteriorly expanded hoxb1a and anteriorly expanded hoxb1b caused by altered RA signaling are patterned by BMP signaling with same temporal dynamics as the normal domains. (A-L) Expression of hoxb1a (bracket) in wild type (A,G) and in Tg(hsp70:chd) embryos subject to HS at the indicated stages (B-F,H-L), without (A-F) and with (G-L) DEAB treatment to inhibit RA signaling. (M-V) Expression of hoxb1b in wild type (M,R) and in Tg(hsp70:chd) embryos subject to HS at the indicated stages (N-Q,S-V), without (M-Q) and with (R-V) RA treatment. DEAB and RA treatments began at shield stage. Embryos are shown at (A-L) 6-somite and (M-V) 90% epiboly stages. Lateral views, dorsal to right. A, n=19/19; B, n=11/11; C, n=10/11; D, n=9/10; E, n=10/12; F, n=16/16; G, n=19/23; H, n=20/24; I, n=19/25; J, n=25/27; K, n=20/23; L, n=21/25; M, n=14/14; N, n=19/22; O, n=19/20; P, n=22/24; Q, n=24/24; R,=20/24; S, n=17/20; T, n=20/23; U, n=21/22; V, n=17/20.

Megumi Hashiguchi, et al. Development. 2013 May 1;140(9):1970-1980.
4.
Fig. 2.

Fig. 2. From: Anteroposterior and dorsoventral patterning are coordinated by an identical patterning clock.

Models of coordinate and independent DV and AP patterning, and anteriorization by FGF inhibition is patterned by BMP signaling with the same temporal dynamics as the normal domain. (A) Models in which DV and AP patterning are coordinated (model 1) or independent (model 2). Embryos are represented at late gastrula stage. The red dashed lines indicate the posteriorly expanded anterior tissue in the top panels and the anteriorly expanded posterior tissue in the bottom panels. (B-K) otx2 expression in anterior neurectoderm in non-heat shocked Tg(hsp70:chd) zebrafish embryos (WT) (B,G) and following HS at the indicated stages (C-F,H-K), with (G-K) or without (B-F) inhibition of FGF signaling by SU5402. The DV width and the AP length of otx2 expression are indicated by horizontal and vertical arrows, respectively. Lateral views, dorsal to right, at 80% epiboly. B, n=30/30; C, n=27/31; D, n=25/29; E, n=21/21; F, n=37/39; G, n=37/39; H, n=34/37; I, n=37/40; J, n=23/25; K, n=34/35.

Megumi Hashiguchi, et al. Development. 2013 May 1;140(9):1970-1980.
5.
Fig. 1.

Fig. 1. From: Anteroposterior and dorsoventral patterning are coordinated by an identical patterning clock.

Progressive DV neurectodermal patterning along the AP axis revealed by a Tg(hsp70:chd) BMP inhibition series. (A-F) six3, (G-L) otx2 and (M-R) gbx1 expression in anterior neurectoderm and (S-X) hoxb1b expression in posterior neurectoderm in non-heat shocked Tg(hsp70:chd) (WT) zebrafish embryos (A,G,M,S) and in Tg(hsp70:chd) embryos subject to heat shock (HS) at the indicated stages to inhibit BMP signaling (B-F,H-L,N-R,T-X). 1K-cell, 1000-cell stage. The DV width of the expression domain is indicated. Lateral views, dorsal to right, except for insets in A and B, which are animal pole views. (A-R) Shown at 80% epiboly stage; (S-X) shown at 90-95% epiboly stage. A, n=9/9; B, n=7/9; C, n=9/10; D, n=11/11; E, n=12/12; F, n=10/10; G, n=30/30; H, n=27/31; I, n=25/29; J, n=21/21; K, n=37/39; L, n=10/10; M, n=9/9; N, n=13/13; O, n=9/9; P, n=8/9; Q, n=9/10; R, n=10/10; S, n=32/32; T, n=26/27; U, n=21/22; V, n=28/29; W, n=17/20; X, n=20/21.

Megumi Hashiguchi, et al. Development. 2013 May 1;140(9):1970-1980.
6.
Fig. 5.

Fig. 5. From: Anteroposterior and dorsoventral patterning are coordinated by an identical patterning clock.

Localization of P-Smad1/5MAPK, P-Smad1/5GSK3 and total Smad1/5 during gastrulation. (A-K) Localization of P-Smad1/5 (A, n=4/4; B, n=5/6), total Smad1/5 (C, n=6/6; D, n=5/5), P-Smad1/5MAPK (E, n=5/5; F, n=4/5; G, n=3/3; H, n=7/7) and P-Smad1/5GSK3 (I, n=3/3; J, n=4/4; K, n=2/3) in sbndtc24 mutants (A,B,E-G,I-K) or wild type (C,D,H) at early gastrula stage (A-C,E-G,I-K), 75% epiboly (D) or 60% epiboly (H) stage. Embryos were injected with hSmad1WT (B,F,J), hSmad1MM (G) or hSmad1GM (K) RNA or were treated with SU5402 (H). (L) Western blot of total Smad5 protein in wild-type embryos and those treated with SU5402 or injected with smad5 MOs. Embryos were collected at 65% epiboly. Actin is a loading control. (M-X) Localization of P-Smad1/5 (M-P), P-Smad1/5MAPK (Q-T) and P-Smad1/5GSK3 (U-X) in wild type at shield (early gastrula) (M, n=8/8; Q, n=10/12; U, n=9/10), 70% epiboly (N, n=6/6; R, n=9/13; V, n=3/5), 80% epiboly (O, n=7/7; S, n=12/15; W, n=4/5) and 90% epiboly (late gastrula) (P, n=7/7; T, n=8/11; X, n=3/5) stage. All are merged confocal images of P-Smad1/5 (green), total Smad1/5 (green), P-Smad1/5MAPK (green), P-Smad1/5GSK3 (green) and DAPI (blue, except red in for C,D). Lateral views, dorsal to right. A, animal pole; Vg, vegetal pole; V, ventral; D, dorsal.

Megumi Hashiguchi, et al. Development. 2013 May 1;140(9):1970-1980.
7.
Fig. 3.

Fig. 3. From: Anteroposterior and dorsoventral patterning are coordinated by an identical patterning clock.

Temporal effectiveness of DMH1 in inhibiting P-Smad1/5, and the temporal patterning dynamics of DV tissues are unchanged when posteriorized or anteriorized by altered Wnt signaling. (A) P-Smad5 western blot for embryos collected at the indicated stages after treatment with the BMP signaling inhibitor DMH1 at the 64-cell, 1000-cell, high and sphere stages. Actin is a loading control. (B-K) Expression of otx2 in untreated wild type (B) and in wild type treated with DMH1 at the indicated stages (C-F). otx2 expression in HS Tg(hsp70:dkk1) embryos without (G) and with DMH1 treatment at the indicated stages (H-K). (L-U) Expression of hoxb1b in Tg(hsp70:chd) embryos not subject to HS (WT) (L,Q) and subject to HS at the indicated stages (M-P,R-U), without (L-P) and with (Q-U) LiCl treatment to activate Wnt signaling and posteriorize the embryos. (V-EE) Expression of six3 (asterisk) and krox20 (arrowheads) in wild type (V,AA) and in Tg(hsp70:chd) embryos subject to HS at the indicated stages (W-Z,BB-EE), without (V-Z) and with (AA-EE) LiCl treatment. The distance from the anterior tip of the head to the anterior boundary of r3 is indicated (V,AA). LiCl treatment was at shield stage. HS of Tg(hsp70:dkk1) embryos was performed at 37°C for 1 hour from 50% epiboly stage (5.3 hpf) to shield stage (6 hpf). Lateral views, dorsal to right. Embryos are shown at (B-K) 80% epiboly, (L-U) 90% epiboly and (V-EE) 6-somite stage. B, n=10/10; C, n=18/19; D, n=17/18; E, n=16/16; F, n=16/16; G, n=16/21; H, n=17/23; I, n=16/22; J, n=18/20; K, n=17/21; L, n=18/18; M, n=16/19; N, n=16/18; O, n=18/19; P, n=16/17; Q, n=9/13; R, n=10/15; S, n=16/18; T, n=18/20; U, n=17/19; V, n=23/23; W, n=18/19; X, n=16/18; Y, n=13/16; Z, n=19/19; AA, n=14/14; BB, n=14/14; CC, n=16/17; DD, n=16/18; EE, n=17/18.

Megumi Hashiguchi, et al. Development. 2013 May 1;140(9):1970-1980.

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