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Expression of connexin genes in the human retina.

Söhl G, Joussen A, Kociok N, Willecke K - BMC Ophthalmol (2010)

Bottom Line: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA.Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina.Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genetik der Universität Bonn, Römerstr, Bonn, Germany. sekretariat@martinus-gymnasium.de

ABSTRACT

Background: Gap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues. Each channel is composed of 12 protein subunits, termed connexins (Cx). In the mouse retina, Cx43 could be localized mostly between astroglial cells whereas expression of Cx36, Cx45 and Cx57 genes has been detected in different neuronal subtypes. In the human retina, however, the expression pattern of connexin genes is largely unknown.

Methods: Northern blot hybridizations, RT-PCR as well as immunofluorescence analyses helped to explore at least partially the expression pattern of the following human connexin genes GJD2 (hCx36), GJC1 (hCx45), GJA9 (hCx59) and GJA10 (hCx62) in the human retina.

Results: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA. Immunofluorescence signals for both connexins could be located in both inner and outer plexiform layer (IPL, OPL). Expression of a third connexin gene denoted as GJA10 (Cx62) was also detected after Northern blot hybridization in the human retina. Interestingly, its gene structure is similar to that of Gja10 (mCx57) being expressed in mouse horizontal cells. RT-PCR analysis suggested that an additional exon of about 25 kb further downstream, coding for 12 amino acid residues, is spliced to the nearly complete reading frame on exon2 of GJA10 (Cx62). Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina.

Conclusion: In contrast to the neuron-expressed connexin genes Gjd2 coding for mCx36, Gjc1 coding for mCx45 and Gja10 coding for mCx57 in the mouse, a subset of 4 connexin genes, including the unique GJA9 (Cx59) and GJA10 (Cx62), could be detected at least as transcript isoforms in the human retina. First immunofluorescence analyses revealed a staining pattern of hCx36 and hCx45 expression both in the IPL and OPL, partially reminiscent to that in the mouse, although additional post-mortem material is needed to further explore their sublamina-specific distribution. Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

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RT-PCR analyses of hCx59 and hCx62 RNAs in different tissues of the human eye. Both the hCx62-specific primer combination USP2 - DSP2 (within the first exon) and the primer combination USP2 - DSP3 (intron spanning) yielded amplicons of about 1.3 kb and 1.1 kb, respectively, only with cDNA from retina. The intron-spanning primer combination USP2-DSP4 even failed to yield a signal with retina cDNA. The amplicon of 550 bp implies that hCx59 seemed to be expressed in retina and also very faintly in lens.
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Figure 3: RT-PCR analyses of hCx59 and hCx62 RNAs in different tissues of the human eye. Both the hCx62-specific primer combination USP2 - DSP2 (within the first exon) and the primer combination USP2 - DSP3 (intron spanning) yielded amplicons of about 1.3 kb and 1.1 kb, respectively, only with cDNA from retina. The intron-spanning primer combination USP2-DSP4 even failed to yield a signal with retina cDNA. The amplicon of 550 bp implies that hCx59 seemed to be expressed in retina and also very faintly in lens.

Mentions: RT-PCR analyses revealed the presence of hCx62 as well as hCx59 transcripts only in cDNA from human retina RNA but not in cDNA from human lens, iris diaphragm, and nerve stump RNA (figure 3). In human retinal cDNA, the unspliced form and the spliced hCx62 transcript isoform, containing exon3 approx. 25 kb downstream of exon2, were detected. However, no amplicon corresponding to the expression of a putative hCx62 transcript isoform containing a third exon about 32 kb downstream of exon 2 (figure 3) was found. A very weak amplicon reflecting hCx59 (GJA9) expression in human lens cDNA is likely due to RNA contamination during tissue preparation (figure 3).


Expression of connexin genes in the human retina.

Söhl G, Joussen A, Kociok N, Willecke K - BMC Ophthalmol (2010)

RT-PCR analyses of hCx59 and hCx62 RNAs in different tissues of the human eye. Both the hCx62-specific primer combination USP2 - DSP2 (within the first exon) and the primer combination USP2 - DSP3 (intron spanning) yielded amplicons of about 1.3 kb and 1.1 kb, respectively, only with cDNA from retina. The intron-spanning primer combination USP2-DSP4 even failed to yield a signal with retina cDNA. The amplicon of 550 bp implies that hCx59 seemed to be expressed in retina and also very faintly in lens.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984586&req=5

Figure 3: RT-PCR analyses of hCx59 and hCx62 RNAs in different tissues of the human eye. Both the hCx62-specific primer combination USP2 - DSP2 (within the first exon) and the primer combination USP2 - DSP3 (intron spanning) yielded amplicons of about 1.3 kb and 1.1 kb, respectively, only with cDNA from retina. The intron-spanning primer combination USP2-DSP4 even failed to yield a signal with retina cDNA. The amplicon of 550 bp implies that hCx59 seemed to be expressed in retina and also very faintly in lens.
Mentions: RT-PCR analyses revealed the presence of hCx62 as well as hCx59 transcripts only in cDNA from human retina RNA but not in cDNA from human lens, iris diaphragm, and nerve stump RNA (figure 3). In human retinal cDNA, the unspliced form and the spliced hCx62 transcript isoform, containing exon3 approx. 25 kb downstream of exon2, were detected. However, no amplicon corresponding to the expression of a putative hCx62 transcript isoform containing a third exon about 32 kb downstream of exon 2 (figure 3) was found. A very weak amplicon reflecting hCx59 (GJA9) expression in human lens cDNA is likely due to RNA contamination during tissue preparation (figure 3).

Bottom Line: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA.Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina.Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genetik der Universität Bonn, Römerstr, Bonn, Germany. sekretariat@martinus-gymnasium.de

ABSTRACT

Background: Gap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues. Each channel is composed of 12 protein subunits, termed connexins (Cx). In the mouse retina, Cx43 could be localized mostly between astroglial cells whereas expression of Cx36, Cx45 and Cx57 genes has been detected in different neuronal subtypes. In the human retina, however, the expression pattern of connexin genes is largely unknown.

Methods: Northern blot hybridizations, RT-PCR as well as immunofluorescence analyses helped to explore at least partially the expression pattern of the following human connexin genes GJD2 (hCx36), GJC1 (hCx45), GJA9 (hCx59) and GJA10 (hCx62) in the human retina.

Results: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA. Immunofluorescence signals for both connexins could be located in both inner and outer plexiform layer (IPL, OPL). Expression of a third connexin gene denoted as GJA10 (Cx62) was also detected after Northern blot hybridization in the human retina. Interestingly, its gene structure is similar to that of Gja10 (mCx57) being expressed in mouse horizontal cells. RT-PCR analysis suggested that an additional exon of about 25 kb further downstream, coding for 12 amino acid residues, is spliced to the nearly complete reading frame on exon2 of GJA10 (Cx62). Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina.

Conclusion: In contrast to the neuron-expressed connexin genes Gjd2 coding for mCx36, Gjc1 coding for mCx45 and Gja10 coding for mCx57 in the mouse, a subset of 4 connexin genes, including the unique GJA9 (Cx59) and GJA10 (Cx62), could be detected at least as transcript isoforms in the human retina. First immunofluorescence analyses revealed a staining pattern of hCx36 and hCx45 expression both in the IPL and OPL, partially reminiscent to that in the mouse, although additional post-mortem material is needed to further explore their sublamina-specific distribution. Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

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