Temporal expression of Cx43.4. (A) Upper panel: Northern blot hybridized with an antisense Cx43.4 RNA probe. 10 μg of total RNA was loaded in each lane for each developmental stage indicated. The mobilities of the 18S (2.1 kb) and the 28S (5.2 kb) rRNAs are indicated at the side. Lower panel: The same membrane was rehybridized with an antisense eF-1α RNA probe as a control for integrity of the mRNA. (B) Western blot of immunoprecipitated Cx43.4 protein from embryos. The first four lanes represent 10 embryos from each stage. The lanes labeled in vitro Cx43.4 and Cx43.4Δct involve immunoprecipitations of proteins translated in vitro from Cx43.4 mRNA and a mutant Cx43.4 mRNA lacking most of the anti-Cx45 epitope region, respectively.

Whole-mount in situ hybridization of Cx43.4 mRNA during oogenesis and gastrulation. (A) Ovary. (B) 1-K cell stage, side view. (C and D) Shield stage, side and vegetal pole views, respectively; dorsal is to the right for both embryos. (E) 85% epiboly; side view with dorsal to the right. (F) 85% epiboly, vegetal pole view with dorsal to the top. (G) Section through the germ ring of an 85% epiboly embryo, dorsal to the right. Dashed lines mark the blastoderm margin in E and F. (H) Section through the germ ring of a 70% epiboly embryo in a more caudal position than in (G); dorsal is to the top. Arrows in E, F, and H point to unlabeled cells in the caudal embryonic shield. The magnification is the same for all panes; scale bar in H, 250 μm.

Whole-mount in situ hybridization of Cx43.4 mRNA during segmentation. (A) 2- to 5-somite stage; dorsal views. Large arrow, tail bud; small arrows, paraxial mesoderm. Scale bar, 250 μm. (B) 2-somite stage; side view. Large arrow, notochord; small arrow, paraxial mesoderm. (C) 5-somite stage; posterior, transverse view at higher magnification. Large arrow, notochord; small arrow, paraxial mesoderm. Scale bar, 100 μm. (D) 10-somite stage, side view, anterior to the left; arrow points to paraxial tissue. (E) Sagital section of a 1-somite embryo; arrow marks notochord. (F) Transverse section of a 2-somite stage embryo, arrow marks expression in paraxial tissue in the tail. Scale bar in F represents 250 μm and is the same for B, D, and E.

Whole-mount in situ hybridization of Cx43.4 mRNA during late segmentation and pharyngula periods. (A) 20-somite stage embryo; side view with anterior to the left. (B) Sagital section through the tail at the 17-somite stage. (C and D) End of segmentation period; side view with anterior to the left. (E and F) Pharyngula period (30 hr); dorsal view with anterior to the left. Arrows point to the floor plate of the central nervous system. Scale bars: D, 250 μm, and the magnification is the same for A and B; E, 250 μm, and the same for C; F, 100 μm.

Cx43.4 protein and Cx43.4 message are localized during gastrulation and early somite stages to similar regions, and the expression of a Cx43.4/GFP fusion protein during blastula stages localizes to regions of cell apposition. (A–D) Immunohistochemical localization of Cx43.4 protein. (A) Tissue from the germ ring of a shield stage embryo; side view with animal pole toward the top; arrow points to blastoderm margin. (B) Tissue from tail bud of a 1-somite stage embryo; dorsal view with posterior to the top; arrow points to blastopore. (C) Tissue from the trunk region of a 1-somite stage embryo; dorsal view with posterior to the bottom; arrow points to the notochord. (D) Scanning confocal microscopy image of the germ ring of a 70% epiboly stage embryo, side view. (E, F) Confocal microscopy images of a living zebrafish embryo after co-injection of Cx43.4/GFP mRNA and rhodamine-dextran at the 1-cell stage. (E) The Cx43.4/GFP fusion protein was visualized during the late blastula period as an extensive array of fluorescent plaques. (F) Superimposed images of the Cx43.4/ GFP protein in E and rhodamine-dextran, showing that most of the fluorescence from the fusion protein is associated with cell surfaces. Scale bar in C represents 100 μm and is the same for A and B; the scale bar in D represents 12 μm and in F, 20 μm.

Whole-mount in situ detection of Cx43.4 mRNA during late gastrulation and tail bud stages in wild-type and ntl mutant embryos. (A and B) Wild-type embryos at 90% and tail bud stage, respectively. (C and D) ntl mutant embryos at the same two stages, respectively. Dorsal views with animal pole or anterior to the left in all panels. Large arrows point to axial hypoblast showing labeled cells in wild-type embryos and an absence of Cx43.4 mRNA detection in ntl embryos, and small arrows in A and C mark the caudal embryonic shield. Scale bar in D represents 250 μm and is the same for the other panels.