Temporal expression of Cx43α transcript and protein. A: Autoradiography of RNase protection analysis of Cx43α mRNA through 120 hr of development. eF-1mRNA was used as a loading control. B: Quantitative analysis of the Cx43α mRNA levels determined by RNase protection. Note that a standard curve was generated using synthetic Cx43α transcripts, with the specific levels of embryonic Cx43α mRNA in picograms being reported on the Y axis. C: Western immunoblot analysis revealing the presence of Cx43α protein in 48 hpf embryos and adult heart and ovary. The mobility of the Cx43α band in the zebrafish samples was not altered by alkaline phosphatase treatment. Lane 1, 48 hours postfertilization (hpf) embryo extract treated with phosphatase inhibitors; lane 2, 48 hpf embryo extract treated with CIP; lane 3, adult zebrafish heart extract treated with phosphatase inhibitors; lane 4, adult zebrafish heart extract treated with CIP; lane 5, adult mouse ovary extract treated with phosphatase inhibitors; lane 6, adult mouse ovary extract treated with CIP; lane 7, adult zebrafish ovary extract treated with phosphatase inhibitors; lane 8, adult zebrafish ovary extract treated with CIP.

Whole-mount in situ hybridization, early stages. A: Animal pole view, 2 hours postfertilization (hpf), 64-cell stage. Maternal transcripts are evident in the deep cells, with little or no Cx43α expression in the enveloping layer. B: Lateral view of embryo shown in A. C: Sectioning of the same embryo confirms the localization of transcripts in deep cells (d). evl, enveloping layer; y, yolk. D: Shield stage (5.5 hpf), animal pole view. Maternal transcript has largely been extinguished. E,F: Anterior (rostral, E) and lateral (F) views of the 16 hpf, 14-somite stage embryo. The most prominent expression at this stage is in the retina. The tail is toward the bottom of these two panels and in G and H. G: Lateral view, 10.7 hpf, tail bud stage. The earliest domain of zygotic Cx43 expression is in the notochord (n, arrows). H: Oblique view, 11.5 hpf, five-somite stage. Peak notochord expression progresses in a rostrocaudal sequence. Same scale as G. Inset: dorsal view of the same embryo. I: Lateral view, 24 hpf embryo. Rostral is to the left in this panel and in J through N. Transcripts are noted in the wall of the trunk dorsal aorta (a) and axial vein (v). Inset: the sprouting intersomitic arteries also demonstrate expression, which extends to the level of the dorsolateral surface of the neural tube. d ao, dorsal aorta. J: Lateral view of the tail, 24 hpf. Notochord (n) expression can be detected in the distal tail. Inset: Near-parasagittal section reveals expression in the rostral (anterior) half of each somite (right inset). For comparison, hybridization with a Cx43 sense riboprobe is shown on the left inset (v, axial vein). K: Higher magnification lateral view, 24 hpf. The axial vein (v) expression appears to sidestep the ventral extent of the blood island (bi). The inset (dorsal view) shows that both dorsal aortae (da, proximal to their coalescence point) express Cx43α. The midbrain-hindbrain boundary region (small, unlabeled arrow) also appears to be a site of expression. L: Near-lateral views, 24 hpf. Left: In addition to the dorsal aortae, transcripts can also be identified in the pronephric ducts (pd). Right: Higher magnification view of the midbrain-hindbrain boundary region shows more clearly that the expression is actually on either side of the midbrain-hindbrain boundary. M,N: Dorsal views of the rostral and caudal embryo, 36 hpf. Cx43α expression is seen in the pronephric primordia (np) bilaterally. The midbrain-hindbrain boundary region continues to show intense expression (unlabeled arrows). a, caudal-most portion of aorta; v, axial vein. Scale bars = 175 μm in A,B,D,F, 160 μm in C,G (applies to H), 220 μm in E, 250 microns in I, 100 μm in J, 280 μm in K, 200 μm in L-N.

Whole-mount in situ hybridization: 48-60 hpf. A: Nearly ventral view, 48 hours postfertilization (hpf). Expression can be seen in tectal ventricle (tv), hypothalamus (h), trigeminal ganglia (Vg), and the forebrain-midbrain boundary (fmb). ot, cardiac outflow tract; pa, pharyngeal arches. B: High-magnification view of the pectoral fin shows predominance of transcripts at the apical ectodermal fold (aef, large arrows), although some expression can also be seen at the base (base, small arrows). C: Near-lateral view, 48 hpf. Intense expression is seen in the fourth ventricle (4th vent) and otic vesicle (ov), whereas comparatively low-level expression is seen in the pectoral fin (pf). D: Near-parasagittal section, 48 hpf. Cx43α is expressed in the pectoral fin bud mesenchyme, pharyngeal arches, and outflow tract (ot) of the heart. E,F: Ventral and lateral views, respectively, of 60 hpf embryo. In addition to the horizontal semicircular canal (hc) in the ear, transcripts are noted in the cardiac outflow tract (ot), ventricle (v), atrioventricular junction, and atrium (a), but not sinus venosus. The shape of this expression domain (arrowheads in F) conforms to that of neural crest (nc) cell migration along the dorsolateral aspects of the looped heart tube. Scale bars = 75 μm in A, 50 μm in B,D, 250 μm in C, 210 μm in E, 240 μm in F.

Whole-mount in situ hybridization: 72-120 hours postfertilization (hpf). A: Ventral view, 72 hpf. Although Cx43α transcripts are difficult to detect in the ear and heart by 72 hpf, they are readily detected in all seven pharyngeal arches (numbered) and in the pectoral fin cartilage (pf). B: Lateral view of the same embryo as in A (pf, pectoral fin). C: Lateral view, 120 hpf. Expression can be seen in the inner nuclear layer (inl) of the retina. D: Oblique dorsal view, 120 hpf. Cx43α expression can be identified in the diencephalon (di), optic tectum (TeO), the hindbrain (hb), and in the boundaries between these regions of the brain, i.e., the forebrain-midbrain boundary (fmb) and the midbrain-hindbrain boundary (mhb). inl, inner nuclear layer; v, ventral retina. E: Oblique dorsal view, 120 hpf. Plane of focus is more dorsal than in D. Cx43α also delineates the rhombomere 1-rhombomere 2 boundary (r1-r2); the region between the midbrain-hindbrain boundary (mhb) and the r1-r2 is the cerebellum. Expression in the fourth ventricle (4th v) persists. Other abbreviations as in D. F: Lateral view, 120 hpf. Cx43α expression in the median fin fold cartilage. Scale bars = 120 μm in A, 125 μm in B, 35 μm in C, 250 μm in D,E, 140 μm in F.

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