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1.
Figure 7.

Figure 7. From: Zic3 is required in the migrating primitive streak for node morphogenesis and left-right patterning.

Zic3 is required in the migrating mesoderm and primitive streak for normal node morphogenesis. (A) and (B) SEM images of a Cre-negative control embryo exhibiting normal node morphology. (C) and (D) SEM images demonstrating abnormal node morphology when Zic3 is deleted from the migrating mesoderm and the primitive streak. (B) and (D) Larger magnification images of (A and C).

Mardi J. Sutherland, et al. Hum Mol Genet. 2013 May 15;22(10):1913-1923.
2.
Figure 4.

Figure 4. From: Zic3 is required in the migrating primitive streak for node morphogenesis and left-right patterning.

Zic3 is required in the migrating mesoderm and primitive streak for normal molecular left–right patterning. (A–D) Images of E8.5 embryos following WISH with Pitx2 (A and B) and Lefty2 (C and D) riboprobes. (A) demonstrates normal left-sided expression of Pitx2 in the control embryo, while the mutant in (B) reveals abnormal, bilateral expression. (C) demonstrates normal left-sided expression of Lefty2 in the control embryo, while the mutant in (D) reveals abnormal, absent expression.

Mardi J. Sutherland, et al. Hum Mol Genet. 2013 May 15;22(10):1913-1923.
3.
Figure 3.

Figure 3. From: Zic3 is required in the migrating primitive streak for node morphogenesis and left-right patterning.

Zic3 is required in the migrating mesoderm and primitive streak for normal heart looping. B) and (D) Representative images of heart defects observed at E10.5 (B) and E13.5 (D) in Zic3del/y;T-Cre+ males. (A) and (C) Stage-matched control embryos. Scale bar is 2 mm. at, atria; ot, outflow tract; lv, left ventricle; rv, right ventricle.

Mardi J. Sutherland, et al. Hum Mol Genet. 2013 May 15;22(10):1913-1923.
4.
Figure 2.

Figure 2. From: Zic3 is required in the migrating primitive streak for node morphogenesis and left-right patterning.

Zic3 is present in the node during the establishment of left–right asymmetry. A transgenic Zic3-LacZ-BAC reporter mouse was generated to monitor Zic3 expression throughout mouse development. (A) and (B) show E7.75 embryos with strong β-gal staining in the node (arrows) and lesser staining in the surrounding tissues. (A) A posterior view of the embryo. (B) A ventral view of the embryo. (C) shows strong β-gal staining throughout an E8.5 embryo but the absence of staining in the heart. (D) A transverse plastic 5 µm section of the β-gal-stained embryo in (C), showing strong staining in the region of the node. The line in (C) indicates the plane of section.

Mardi J. Sutherland, et al. Hum Mol Genet. 2013 May 15;22(10):1913-1923.
5.
Figure 6.

Figure 6. From: Zic3 is required in the migrating primitive streak for node morphogenesis and left-right patterning.

Zic3 is required for normal node morphogenesis. (A) and (B) show SEM of node ultrastructure in WT (A) and Zic3 null embryos (B). The node of WT embryos is a distinct triangular-shaped depression (A). The node of Zic3 mutant embryos is disorganized and interspersed with nests of larger endoderm cells (B). (C) and (D) Images of IHC performed using anti-acetylated tubulin (green) as a marker of cilia. (E) and (F) Images of IHC performed with tight junction marker anti-ZO-1 (green) and anti-acetylated tubulin (red). (G) and (H) SEM images of cilia on the node cells. Zic3 null cilia (H) were significantly shorter compared with WT (G and I). (I) Evaluation of node cells performed on WT and Zic3 null embryos after IHC.

Mardi J. Sutherland, et al. Hum Mol Genet. 2013 May 15;22(10):1913-1923.
6.
Figure 1.

Figure 1. From: Zic3 is required in the migrating primitive streak for node morphogenesis and left-right patterning.

Epiblast deletion of Zic3 recapitulates the Zic3 null phenotype. (A) A schematic of the Zic3 conditional allele. Targeted insertion of a neomycin cassette in intron 1 of mouse Zic3 (top) allowed for selection for recombination. Removal of the neomycin cassette resulted in the Zic3 floxed allele (middle) in which loxP sites flank exon 1 of Zic3. Exon 1 is removed from a tissue of interest (bottom) when the Zic3 floxed mouse is mated with a mouse carrying a tissue-specific Cre recombinase. LoxP sites are shown in red and FRT sites are shown in blue. To verify proper recombination of the floxed Zic3 allele, Zic3flox/flox females were mated with Sox2-Cre males. (B) shows a loss of Zic3 mRNA by WISH in Zic3del/y;Sox2-Cre+ embryos when compared with control littermates (C). (D) shows normal heart looping in a Cre-negative male. Zic3del/y;Sox2-Cre+ embryos had a similar phenotype to Zic3 null embryos including heart (E) and neural tube (F) defects.

Mardi J. Sutherland, et al. Hum Mol Genet. 2013 May 15;22(10):1913-1923.
7.
Figure 5.

Figure 5. From: Zic3 is required in the migrating primitive streak for node morphogenesis and left-right patterning.

Zic3 RNA deficiency in node cells following gastrulation does not alter left–right patterning. (A)–(D) show WISH of E8.5 embryos using an antisense riboprobe for Pitx2 expression. Zic3 mutants (B) and (D) and their control littermates (A and C) display normal Pitx2 expression in the left LPM. (E)–(H) E9.5 embryos with proper rightward looping of the previously linear heart tube. (I) A representative image of live EGFP cell enrichment from node regions using the BD FACS Aria sorter. The inset shows a representative image of strong, specific live EGFP expression in the node of an E7.75 embryo. The node and embryo are outlined. (J) shows RT–PCR results of RNA isolated from node cells from 0 to 4 somite stage Zic3del/y;FOXJ1-Cre+; FOXJ1-EGFP (del/y), Zic3flox/y; FOXJ1-Cre-:FOXJ1-EGFP (flox/y) and FOXJ1-EGFP (WT GFP+) embryos. EGFP-negative cells from a pool of FOXJ1-EGFP embryos were sorted and analyzed (WT GFP−). Zic3 expression is absent in Del/y node cells, while the rest of the pooled node cells shows strong Zic3 expression. Gapdh was used as a positive control. The negative control was a mastermix of PCR reagents with no cDNA.

Mardi J. Sutherland, et al. Hum Mol Genet. 2013 May 15;22(10):1913-1923.

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