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First published online 5 October 2004
doi: 10.1242/jcs.01413

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The novel mouse connexin39 gene is expressed in developing striated muscle fibers

Institut für Genetik, Abteilung Molekulargenetik, Universität Bonn, Römerstr. 164, 53117 Bonn, Germany

View larger version (53K): [in a new window]   Fig. 1. Genomic locus of mCx39 containing the unspliced open reading frame. Predicted transmembrane regions (HUSAR subprogram Tmhmm) are underlined and the boxed peptide sequences represent the epitopes to which antisera were raised. Conserved cysteine residues are encircled. Underlined nucleotide sequences represent (from upstream to downstream) the Kozak-consensus motif of translational initiation, both the splice-donor and acceptor sites and the canonical motifs for transcriptional termination. Conserved nucleotides are shown in bold, nucleotide stretches rich in pyrimidines are italic and the branch point of splicing is boxed. The sequence of the 1.5 kb intron between exons 1 and 2 is largely excluded. After splicing, both shaded glycine residues are replaced by one glycine encoded by the restored triplet. Nucleotides and amino acids in bold italics represent the following exchanges found between the 129Sv-derived mCx39 sequence (AJ414562) and the C57BL/6-derived version: nucleotide 2291 G to A; 2546 T to C; 2580 G to T; leading to a Gly to Cys substitution at nucleotide 345 and C to T substitution at nucleotide 2613. The genomic structure of mouse and human connexin genes whose coding region is restored after functional splicing as illustrated in the diagram.

View larger version (64K): [in a new window]   Fig. 2. Alignment of amino acid sequences of mCx39 and its putative orthologue hCx40.1. Potential cAMP-, PKC- and casein kinase II (CKII)-phosphorylation sites within the cytoplasmic loop and C-terminus are indicated in bold italics and those within extracellular or transmembrane domains were omitted, owing to their inaccessibility to kinases. In mCx39, one cAMP (position 203; R/K-X-X-S/T), three PKC (positions 170, 203 and 300; S/T-X-R/K) and six CKII (positions 190, 236, 290, 296, 310 and 319; S/T-X-X-D/E) phosphorylation sites were found. In hCx40.1, two cAMP (positions 177 and 282), six PKC (pos. 195, 235, 236, 277, 281 and 303) and four CKII (pos. 151, 251, 301 and 325) phosphorylation sites were found. Putative transmembrane regions are boxed. Asterisks, conserved residues; double dots, similar residues; single dots, like residues. Note that both sequences display gaps when aligned by the algorithms of the HUSAR-derived program `sequence pair comparison'. The nucleotide/protein sequences of both connexins have been deposited in the GenBank/EMBL/DDBJ databases under accession numbers AJ414562 (mCx39) and AJ414564 (hCx40.1).

View larger version (81K): [in a new window]   Fig. 3. Expression of Cx39 mRNA in different mouse tissues detected by northern blot hybridization. (A,B) Similar amounts of total RNA were applied as demonstrated after staining of the 18S rRNA with ethidium bromide. (A) Expression of mCx39 mRNA during different developmental stages (ED13.5 to P0) of the embryo but not in yolk sac, placenta or in utero. (B) In the eye and chest (including intercostal muscle) of neonatal (P0) and two-day-old mice (P2) as well as in the P2 heads after removing the brain, Cx39 mRNA was expressed at low levels.

View larger version (42K): [in a new window]   Fig. 4. RT-PCR analysis of mCx39 expression in different tissues and of different connexins in the diaphragm of the mouse. (A,B) The presence of mCx39 mRNA in various embryonic (ED16.5) and neonatal (P0) tissues of mice is confirmed by using an intron-spanning primer combination to amplify a 1.1 kb product. Amplification of a 243 bp product specific for ß-actin cDNA (De Sousa et al., 1993) demonstrated the quality of the reverse-transcribed cDNA except for neonatal eye. (C) RT-PCR analysis of cDNA from neonatal (P0) diaphragm revealed additional transcription of Cx26, Cx32, Cx37, Cx40 and Cx45 in this tissue (upper gel). PCR with genomic DNA displayed the appropriately sized products of the tested connexins (lower gel).

View larger version (12K): [in a new window]   Fig. 5. Immunoblot analysis of mCx39-transfected HeLa cells and different mouse tissues. (A) Two signals at about 40 kDa and 80 kDa were prominent in mCx39-transfected HeLa cells but not in wild-type cells. (B-D) Expression of Cx39 in different mouse tissues at different stages of development. At ED16.5 (B) and P0 (C), both signals at 40 and 80 kDa could be readily detected but were absent after probing various adult mouse tissues (D). Thus, the 80 kDa protein band is suspected to be the dimeric form of Cx39.

View larger version (92K): [in a new window]   Fig. 6. Immunolocalization of Cx39 in different developing mouse striated muscles. (A-G) Affinity-purified polyclonal antibodies directed to the C-terminus of mCx39 were used to label 10 µm cryosections of the diaphragm (A-D), the intercostal muscle (E, F) and the hind limb (G). Punctate green staining between myotubes (A,C,E-G) indicated expression of mCx39, which was absent when primary antibodies were omitted (B,D). (H) Immunofluorescence analysis of cultured HeLa cells stably transfected with the coding DNA of mCx39 after incubation with rabbit anti-Cx39 and Alexa488-conjugated goat anti-rabbit immunoglobulin. HeLa mCx39 transfectants showed the expected connexin-specific punctate staining pattern (green) on contacting plasma membranes. Nuclei were counterstained in red with propidium iodide. Bar, 20 µm.

View larger version (55K): [in a new window]   Fig. 9. Whole tissue mounts of neonatal mouse diaphragm. (A) Whole diaphragm from a neonatal mouse immunostained for Cx39. (B) Dye spreading of Alexa488 into neonatal diaphragm 10 minutes after microinjection. (C) Staining of a single myotube of the neonatal diaphragm 10 minutes after injection of rhodamine dextran. Injected cells are marked with an arrow. Bar, 20 µm.

View larger version (105K): [in a new window]   Fig. 7. Immunolocalization of various connexins in embryonic and neonatal diaphragm. (A-C) Expression of Cx26 (A) and Cx32 (B) is restricted to the developing liver and Cx37 to the endothelium (C). (D) Expression of Cx40 was detected in blood vessels. (E) Cx43 localized near the edge of the diaphragm. (F) Expression of the Cx45 gene, indicated by ß-gal staining in tissue from Cx45 (LacZ+) mice (Krüger et al., 2000), is confined to the neonatal lung (shown in the upper part above the dotted line) and to smooth muscle cells around blood vessels (Krüger et al., 2000) of the neonatal diaphragm (lower part). Bar, 20 µm.

View larger version (44K): [in a new window]   Fig. 8. Immunolocalization of Cx39 in intercostal muscle and diaphragm of embryonic mice. (A-C) 10 µm cryosections of embryonic (ED14.5) intercostal muscle (A) and diaphragm (B,C) were incubated with rabbit anti-Cx39 and Alexa488-conjugated goat anti-rabbit antibodies. The punctate green staining pattern suggests that the expression of Cx39 at ED14.5 occurs only in primary myotubes. (C) Diaphragm after incubation without anti-Cx39 antibodies. Bar, 20 µm.

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