Levin M and Mercola M (1998), Gap junctions are involved in the early generat...

XB-ART-14011

Dev Biol 1998 Nov 01;2031:90-105. doi: 10.1006/dbio.1998.9024.

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Gap junctions are involved in the early generation of left-right asymmetry.

Levin M , Mercola M .

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Invariant left-right asymmetry of the visceral organs is a fundamental feature of vertebrate embryogenesis. While a cascade of asymmetrically expressed genes has been described, the embryonic mechanism that orients the left-right axis relative to the dorsoventral and anteroposterior axes (a prerequisite for asymmetric gene expression) is unknown. We propose that this process involves dorsoventral differences in cell-cell communication through gap junctions composed of connexin proteins. Global modulation of gap junctional states in Xenopus embryos by pharmacological agents specifically induced heterotaxia involving mirror-image reversals of heart, gut, and gall bladder. Greatest sensitivity was observed between st. 5 and st. 12, well before the onset of organogenesis. Moreover, heterotaxia was also induced following microinjection of dominant negative and wild-type connexin mRNAs to modify the endogenous dorsoventral difference in junctional communication. Heterotaxia was induced by either blocking gap junction communication (GJC) dorsally or by introducing communication ventrally (but not the reverse). Both connexin misexpression and exposure to GJC-modifying drugs altered expression of the normally left-sided gene XNR-1, demonstrating that GJC functions upstream of XNR-1 in the pathway that patterns left-right asymmetry. Finally, lineage analysis to follow the progeny of microinjected cells indicated that they generally do not contribute the asymmetric organs. Together with the early sensitivity window, this suggests that GJC functions as part of a fundamental, early aspect of left-right patterning. In addition, we show that a potential regulatory mutation in Connexin43 is sufficient to cause heterotaxia. Despite uncertainty about the prevalence of the serine364 to proline substitution reported in human patients with laterality defects, the mutant protein is both a mild hypomorph and a potent antimorph as determined by the effect of its expression on left-right patterning. Taken together, our data suggest that endogenous dorsoventral differences in GJC within the early embryo are needed to consistently orient left-right asymmetry.

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Species referenced: Xenopus
Genes referenced: gja1 gjb2 nodal1

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	FIG. 3. Examples of laterality phenotypes induced by manipulation of GJC. All embryos are shown in ventral views (thus the embryo right is the reader left), with anterior toward the top. (A) A control stage 45 embryo. The heart loops toward the embryo right, the axis of gut coiling points toward the left, and the gall bladder is on the right. (B) Embryos treated as in Figs. 2 and 4 that exhibited aberrant sidedness in any one of these three organs were scored as heterotaxic: inverted heart (B), inverted gut and gall bladder (C), and inverted heart, gut, and gall bladder (D). The clear hearts are difficult to photograph in living specimens (A); therefore, for clarity, staining with the MF20 antibody is shown in normal (E) and situs inversus totalis (F) embryos. Red arrowheads indicate heart loops; yellow arrowheads indicate gut loops; green arrowheads indicate gall bladders. Direction of the arrowhead indicates situs.
	FIG. 6. Lineage analysis of injected cells shows that they do not contribute to asymmetric organs. Embryos were injected with synthetic mRNAs (as in Fig. 4) and developed for 􏰁-galactosidase expression. Nuclear blue stain (indicated with green arrowheads) indicates cells which received injected mRNA. Dorsal injections (A) usually result in expression in the notochord and neural tube, and the dorsal halves of somites (green arrowheads), with an occasional contribution to the heart. Ventral injections (D) result in expression in the ventral half of somites (green arrowheads), with occasional stain in the gut. In more than 80% of the embryos with reversed organ situs, no 􏰁-galactosidase stain was observed in the heart, stomach, or gall bladder (e.g., F and G, compare to E). This determination was made in embryos whose hearts were unstained with MF20 antibody, to ensure that any 􏰁-galactosidase expression would be detected and not masked by red stain.
	FIG. 7. GJC is upstream of XNR-1 expression. Embryos were injected with synthetic mRNAs (as in Fig. 4) or exposed to drugs until st. 20 (as in Fig. 2), allowed to develop to st. 214, fixed, and processed for in situ hybridization with a probe for XNR-1. In 66% of wild-type embryos, stain is seen in the left flank (A), but not in the right (B). C shows the same in section. Double-sided (D) and right-sided (G) stain was not observed (n 􏰇 159). Stain could not be detected in 34% of the embryos (J). In contrast, embryos injected with Cx26 mRNA showed incidences of 52, 1, 46, and 1% of left-sided, right-sided, absent, and bilateral expression, respectively (n 􏰇 109). This is statistically different from the control embryos to 􏰯2 􏰇 4.55, P 􏰇 0.03. Embryos injected with Cx43 exhibited 68, 0, 27, and 5% of left-sided, right-sided, absent, and bilateral expression, respectively (n 􏰇 78). This effect was not statistically significant (P 􏰇 0.8). H7 injections resulted in 43, 5, 38, and 14% of left-sided, right-sided, absent, and bilateral expression, respectively (n 􏰇 42). This distribution of phenotypes is statistically different from the control embryos to 􏰯2 􏰇 6.57, P 􏰇 0.01. Exposure to anandamide resulted in 47, 1, 49, and 3% of left-sided, right-sided, absent, and bilateral expression, respectively (n 􏰇 37). This is statistically different from the control embryos to 􏰯2 􏰇 7.1, P 􏰇 0.008. Exposure to heptanol resulted in 19, 0, 81, and 0% of left-sided, right-sided, absent, and bilateral expression, respectively (n 􏰇 37). This distribution is different from the control to 􏰯2 􏰇 25.32, P 􏰇 4.85 􏰭 10􏰄7. Glycyrrhetinic acid exposure resulted in 10, 2, 86, and 2% of left-sided, right-sided, absent, and bilateral expression, respectively (n 􏰇 59). This distribution is different from the control to 􏰯2 􏰇 51.53, P 􏰇 7.05 􏰭 10􏰄13. In A, red arrowheads indicate XNR-1 expression domains, white arrowheads indicate lack of domain, and yellow letters and indicate left- and right-side views. M summarizes these numerical data.