Connexin 48.5 Is Required for Normal Cardiovascular Function and Lens Development in Zebrafish Embryos

Abstract

Gap junctions are composed of connexin (Cx) proteins and mediate intercellular communication required for many developmental and physiological processes. Here we describe the isolation and characterization of Cx48.5, a zebrafish connexin with the highest sequence identity to mammalian Cx46. Expression analysis showed that Cx48.5 is expressed in the adult and embryonic lens and heart, adult testis, and transiently in the embryonic otic vesicles. Injection of Cx48.5 cRNA into Xenopus oocytes elicited intercellular electrical coupling with voltage sensitivity similar to mammalian Cx46. In single oocytes, Cx48.5 also induced large outward currents on depolarization, consistent with gap-junctional hemichannels. Disruption of Cx48.5 expression in embryos with antisense morpholino oligos (morpholinos) revealed that Cx48.5 has an essential role in the maintenance of lens homeostasis. The morpholino-treated embryos also developed small lenses and eyes as well as severe cardiovascular abnormalities.Gap junctions are clusters of intercellular channels that allow the propagation of small metabolites, signaling molecules, and electrical impulses between adjacent cells. The protein units that make up the gap junction channels in vertebrates are called connexins and are coded for by a multigene family with 20 or more members in mammals (1). Gap junctions play vital roles in the functions of virtually all organs, including the lens and the heart. The evidence to date suggests that distinct sets of connexins may play a role in the development and function of organs like the lens and the heart. Three connexins are expressed in the mammalian lens. Connexin 43 is expressed in the undifferentiated lens epithelium, whereas Cx46 1 and Cx50 are typically considered lens-specific and are predominantly expressed in the lens fiber cells (for review, see Ref.2). Both Cx46 and Cx50 knock-out mice developed cataracts (3-5), and the Cx50 knock-out mice also developed small lenses and small eyes.Four connexins are abundantly expressed in the mammalian heart: Cx43, Cx40, Cx45, and Cx37. Connexin 43 is expressed in the atrial and ventricular working myocardium, Cx40 is found in the atrial working myocardium and the conducting bundle branches and Purkinje fibers, whereas Cx45 is restricted to the atrioventricular conduction system, and Cx37 is restricted to endothelial and endocardial cells (for reviews, see Refs. 6 -8). Mouse knock-out experiments have demonstrated that Cx43, Cx40, and Cx45 are all essential for cardiac development and function. For instance, connexin 43 knock-out mice died neonatally from pulmonary outflow tract obstruction (9). Connexin 40-deficient mice on the other hand exhibited slowed cardiac conduction and a partial atrioventricular block (10, 11). Last, Cx45 knock-out mice died by embryonic day 10 displaying the effects of a conduction block, endocardial cushion defects, and abnormalities of vascular development (12, 13). The Cx37 knock-out mice had no apparent cardiovascular abnormalities (14). Although the expression a...

A novel mutation impairing the tertiary structure and stability of γC-crystallin (CRYGC) leads to cataract formation in humans and zebrafish lens

“…Previous work demonstrated that Cx50 is required for the development of normal lens and eye size in mice [White et al, 1998]. Zebrafish embryos treated with antisense morpholino oligos against Cx48.5 (an ortholog of mammalian Cx46) developed small lenses and eyes [Cheng et al, 2004]. Thus, the downstream changes caused by p.Gly129Cys mutant, such as decreased connexin proteins, may partly account for small lens and eyes phenotype in embryos overexpressing γC-crystallin p.Gly129Cys mutant.…”

Connexin 39.9 Protein Is Necessary for Coordinated Activation of Slow-twitch Muscle and Normal Behavior in Zebrafish

“…In mice, Cx46 is expressed in the lens and heart, and Cx46-deficient mice develop cataracts, indicating that Cx46-dependent gap junctional communication is essential for differentiation of the lens (59,60). Likewise, zebrafish cx48.5 is expressed in the lens and heart, and antisense knockdown of Cx48.5 causes a developmental defect of the lens that is similar to cataracts, suggesting that zebrafish Cx48.5 is the functional counterpart of mammalian Cx46 (61,62). On the other hand, cx39.9 is expressed in the skeletal muscle as well as in the lens, signifying that Cx39.9 has diverged from the GJA3 connexin subfamily.…”

Cardiac conduction is required to preserve cardiac chamber morphology

“…To understand the molecular nature of the dco phenotype, we genetically mapped the dco s226 mutation to a 0.33cM region on chromosome 9 and identified a mutation in the connexin48.5 (cx48.5) gene (9) (Fig. 1 D and E).…”