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Dev Dyn
2005 Jul 01;2333:864-71. doi: 10.1002/dvdy.20420.
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Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevis.
De Boer TP
,
Kok B
,
Neuteboom KI
,
Spieker N
,
De Graaf J
,
Destrée OH
,
Rook MB
,
Van Veen TA
,
Jongsma HJ
,
Vos MA
,
De Bakker JM
,
Van Der Heyden MA
.
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Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (approximately 62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals.
Figure 4. XlCx40.4 expression in the developing Xenopus embryos is present in the presomitic paraxial mesoderm and somites. Whole-mount in situ hybridization was performed on stage 19/20, stage 25/26, and stage 35/36 embryos. Upper left panel: anterior view; signal is visible in the first somites (arrows); dorsal (D) and ventral (V) sides of the embryo are indicated. Upper right panel: dorsoposterior view; signal is visible in the first somites/presomitic paraxial mesoderm (arrows); dorsal (D) and ventral (V) sides of the embryo are indicated. Middle and lower panels: rightlateral view; signal is visible in the somites. No signals were found in sense negative controls (inserts).
mgc69466 (MGC69466 protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anteriorright, dorsal up [insert is antisense control],
(note authors staged this embryo at 35/36 but only control looks this old).
Fig. 2. Cladogram of Xenopus laevis Cx [connexin] sequences. The nucleotide sequences of the protein coding region of 8 Cxs were analyzed using the ClustalW method of the Megalign program of the Lasergene software package DNASTAR. The scale beneath the tree measures the evolutionary distance between the sequences, and units indicate the number of substitution events.
[note: the sequence for clone XlCx40.4 BLASTS to gene: mgc69466]
Fig. 3. Developmental and organ expression of XlCx40.4. A: Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis of X. laevis total RNA at different stages of development. Nieuwkoop and Faber stages are indicated at the top. B: RT‐PCR analysis of adult X. laevis organ and tissue RNA. PCR with histone H4 was used as loading control.
Fig. 4. A,B: Localization of ectopically expressed XlCx40.4‐HcRed fusion protein in HEK293 cells in two independent transient transfections. Arrows indicate typical gap junctional distribution of HcRed‐tagged fusion protein. C,D: Corresponding transmission images are shown.
Fig. 6. Electrophysiological evaluation of XlCx40.4 gap junction channels in N2A cells. A: Time‐dependent inactivation of junctional coupling in response to transjunctional potentials (control). Inhibition of junctional coupling by halothane (halothane). B: Relationship between gap junction steady‐state conductance and Vj. Mean normalized steady state conductance ± SEM (gss/ginst) of eight independent recordings plotted against Vj matches a two‐state Boltzmann distribution.
