Zebrafish connexin 79.8 (Gja8a): A lens connexin used as an electrical synapse in some neurons

Abstract

In the mammalian central nervous system a remarkably small number of connexins is used in electrical synapses, with the majority formed from Cx36. A larger number has been detected in teleosts, with some seeming to serve restricted roles. Here we report the discovery of a new connexin expressed in the zebrafish lens and a limited set of neurons. Zebrafish cx79.8 (gja8a), previously annotated incorrectly as cx50.5 based on a partial cDNA sequence, is a homologue of mammalian Cx50 (Gja8). We examined its expression through transgenic promoter-reporter constructs, in situ hybridization, and immunolabeling, and examined regulation of coupling in transfected HeLa cells. cx79.8 was expressed most strongly in the lens, but expression was also found in several groups of neurons in the cerebellum and related areas at the midbrain-hindbrain boundary, in cone photoreceptors, and in neurons in the retinal inner nuclear and ganglion cell layers. Labeling in the retina with antibodies against two C-terminal epitopes revealed numerous small punctate spots in the inner plexiform layer and along the somata of cones. Abundant gap junctions were labeled in the outer 1/3 of the lens, but were absent from the center, suggesting that the epitopes or the entire protein was absent from the center. Cx79.8 tracer coupling was strongly regulated by phosphorylation, and was extremely low in control conditions in HeLa cells due to protein phosphatase 2A activity. These properties allow coupling to be strongly restricted in situ, a frequently observed property for electrical synapses.

Connexinplexity: The spatial and temporal expression of connexin genes during vertebrate organogenesis

“…and gja10b/Cx52.6 in presumptive horizontal cells, all well-matching published reports on the expression of these genes 64,99,111,114,115 (Supp. Figure 5; Supp.…”

“…We also find expected patterns for connexins that have well-known, spatially-restricted expression. For example, gja8b/Cx44.1 is expressed almost exclusively in the early developing lens [111][112][113][114] , and in the scRNA-seq dataset we find expression of gja8b/Cx44.1 within clusters with transcriptional profiles consistent with lens cells (Fig. 1Biii; Supp.…”

Loss of Gap Junction Delta-2 (GJD2) gene orthologs leads to refractive error in zebrafish

“…6q–t ) mutants. To rule out that the MAB3045 antibody cross-reacted with other related retinal connexins, i.e., Cx34.1 ( gjd1a ) and Cx34.7 ( gjd1b ) 29 , 32 , 34 , 49 , 51 , we examined the staining profile in a mutant for both gjd2 paralogues ( gjd2a; gjd2b double mutant). A complete loss of immunostaining in the gjd2a; gjd2b double mutant (Supplementary Fig.…”

“…Consistent with our results in the gjd2b (Cx35.1) mutant, the loss of mammalian Cx46 and Cx50 also leads to cataract development 90 – 93 , and mutations in human Cx46 and Cx50 have been associated with autosomal dominant congenital cataract 94 – 97 . Although to date, Cx35.1 ( gjd2b ) has not been described as a lenticular connexin, four other main connexins, Cx43, Cx44.1, Cx48.5 98 , 99 , and the larger Cx79.8 29 , have previously been found in the lens. Whereas Cx48.5 zebrafish morphants showed modest nuclear opacities at 9.5 dpf 99 , the gjd2b (Cx35.1) mutant showed a cataract phenotype in a much later developmental stage (60% at 3 mpf).…”

Localized Calcium Signaling and the Control of Coupling at Cx36 Gap Junctions

“…Furthermore, the CaMKII activity in HeLa cells was not constitutive. The intervention routinely performed in our group of inhibiting PKA activity with Rp-8-cpt-cAMPS, which appears to increase coupling by reducing PKAstimulated Protein Phosphatase 2A activity (Kothmann et al, 2009;Yoshikawa et al, 2017), increased coupling in a manner independent of CaMKII activity. Thus, coupling could be increased by constitutive activity of an as yet unidentified protein kinase as well as through glutamate-inducible CaMKII activity.…”