Submitted on August 21, 2003
Accepted on December 23, 2003
Molecular Cloning, Functional Analysis, and RNA Expression Analysis of Connexin45.6: a Zebrafish Cardiovascular Connexin
Tara L. Christie1, Rickie Mui2, Thomas W. White2, and Gunnar Valdimarsson3*
1 Department of Zoology, University of Manitoba, Winnipeg, Manitoba, Canada
2 Department of Physiology and Biophysics, SUNY, Stony Brook, New York, USA
3 Department of Zoology, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Biochemistry and Medical Genetics, University of Manitoba, Stony Brook, New York, USA
* To whom correspondence should be addressed. E-mail: valdimar{at}cc.umanitoba.ca.
In the vertebrate cardiovascular system, gap junctions function in intercellular communication essential for both the coordinated propagation of the heartbeat, and the control of vasomotor responses in the vascular system. Connexins, the protein subunits of gap junctions, are coded for by a multigene family. In this study, a connexin gene (zfCx45.6) which exhibits 53% amino acid identity to chick Cx42 was cloned from zebrafish genomic DNA. With the use of the LN54 radiation hybrid panel, zfCx45.6 was mapped to zebrafish linkage group 9. Northern blots and RT-PCR revealed the presence of zfCx45.6 mRNA in the embryo before 2 hours post fertilization (hpf), and then again beginning at about 12 hpf, after which time no major changes in relative expression levels were detected. In the adult, zfCx45.6 mRNA continued to be detected in the heart, as well as the brain, liver and ovary, but not the lens. Whole mount in-situ hybridization revealed zfCx45.6 mRNA was expressed at high levels in the major vessels of the entire embryo, and both the atrium and ventricle of the adult heart. Expression of zfCx45.6 channels in paired Xenopus oocytes produced high levels of intercellular coupling that was voltage-sensitive. With the previous isolation of zebrafish Cx43 and Cx43.4, zebrafish orthologues have now been isolated for three of the four connexins expressed in the mammalian cardiovascular system.
