Cloning, embryonic expression, and functional characterization of two novel connexins from Xenopus laevis q,qq Teun P. de Boer a,1 , Bart Kok a,1 , Giulietta Roe ¨l b , Toon A.B. van Veen a , Olivier H.J. Destre ´e b , Martin B. Rook a , Marc A. Vos a , Jacques M.T. de Bakker a , Marcel A.G. van der Heyden a, * a Department of Medical Physiology, HLCU, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands b Hubrecht laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands Received 11 August 2006 Available online 31 August 2006 Abstract Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays >60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell–cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Connexin; Phylogeny; Gap junction; Xenopus; Expression; Electrophysiology; Organizer; Pronephros; Endoderm; Mesoderm; Cement gland; Liver anlage Gap junctions are aggregates of intercellular communi- cation channels connecting the cytoplasm of adjacent cells. Multicellular animal species have acquired gap-junctions to enable the direct exchange of small signaling molecules, metabolites, and ions between cells. Invertebrates mainly use innexin proteins to constitute their gap-junctions [1]. Chordate species, however, make use of a sequence unre- lated class of proteins named connexins (Cxs) as gap-junc- tion building blocks [2]. Recently, a third class of gap- junction proteins was exposed, called pannexins [3]. It became clear that pannexins are expressed in both verte- brates and non-vertebrates. Genome screens demonstrated, however, the presence of only three different pannexins, in contrast to the great numbers of innexins and Cxs. There- fore, although speculative, the different Cxs probably have evolved to encounter the specific requirements of cell–cell communication in more complex organisms, in addition to the pannexins. To form a gap-junction, a hexamer of innexins or Cxs, the latter is named a connexon, docks at a similar cluster on an adjacent cell thereby forming a functional intercellu- lar channel [4]. On the other hand, connexons by themselves may form functional hemichannels in non-junc- tional plasmamembranes, which, among other functions, are thought to be involved in osmoregulation [5]. 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.08.121 q DNA sequences in this report have been deposited at GenBank (Accession Nos.: DQ180493 (Cx28.6) and DQ180492 (Cx29)). qq Abbreviations: Cx, connexin; ORF, open reading frame; EST, expressed sequence tag; WMISH, whole-mount in situ hybridization. * Corresponding author. Fax: +31 30 2539036. E-mail address: m.a.g.vanderheyden@med.uu.nl (M.A.G. van der Heyden). 1 These authors contributed equally to this work. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 349 (2006) 855–862 BBRC