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In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.

Background: Gene silencing in the retina using RNA interference could open broad possibilities for functional studies of genes in vivo and for therapeutic interventions in eye disorders. Therefore, there is a considerable demand for protocols to deliver siRNA into the vertebrate retina. In this work we explored a possibility to deliver synthetic 21 bp siRNA into the mouse retina after intravitreal application using a non-viral carrier., Methods: Fluorescently labelled synthetic 21 bp siRNA duplex was combined with Transit-TKO transfection reagent and injected intravitreally into adult mice eyes. Eyes cryostat sections and whole mount retinas were prepared 24-48 h post-injection, stained with either Hoechst 33342 (cell nuclei) or immunostained with anti-GFAP antibody (astroglia cells marker). Distribution of fluorescent siRNA signal in the retina was investigated., Results: Single intravitreal injection of as little as 5 ng of siRNA combined with Transit-TKO transfection reagent by a modified protocol provided robust and non-toxic delivery of the siRNA into the retina. However, siRNA accumulation was predominantly confined to ganglion cells layer as analysed 24 h post-injection. Furthermore, siRNA containing particles were localized along GFAP cytoskeleton of retinal astroglial cells hinting on intracellular localization of the siRNA CONCLUSIONS: In this work we demonstrated that siRNA can be efficiently delivered into the vertebrate retina in vivo with low-toxicity using a non-viral carrier, specifically Transit-TKO transfection reagent. However, the capacity of siRNA delivered by our protocol to induce gene silencing in the retina has to be further evaluated. Our report could raise a closer look on Transit-TKO transfection reagent as a promising siRNA carrier in vivo and be of interest for the researchers and companies who work on development of ocular RNAi techniques.

Gap junctions are among the most widely distributed cell structures involved in cell-to-cell communication. Recently completed genome sequencing projects including species from all major phyla have demonstrated the existence of three distinct gene families, the connexins, pannexins, and innexins, as molecular building blocks of gap junctional communication. In the present study, the authors have addressed the molecular complexity of gap junction gene expression in the zebrafish retina, a remarkably complex sensory organ built by diverse neuronal subtypes. Using a combination of cDNA library and genomic DNA library screening and/or RACE technology, the authors have cloned, in addition to the four previously reported connexins, seven novel connexins and four pannexin transcripts resembling two pannexin genes. This result demonstrates the presence of two distinct gap junction type gene families and indicates a remarkable molecular and functional diversity of gap junction-mediated coupling in the fish retina.

Receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including cell proliferation, migration and differentiation. Here we investigated potential roles of RPTPs in the developing mouse retina. Using a degenerate oligonucleotide-based reverse transcription polymerase chain reaction approach, we identified 11 different RPTPs in the retina at embryonic stage 13 (E13). Subsequently, the expression patterns of RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma in the retina from embryonic stages to adult were analyzed in detail using quantitative real-time-PCR, in situ hybridization, immunohistochemistry and Western blotting. At E13, all six RPTPs are expressed in actively cycling retinal progenitor cells and postmitotic newborn retinal neurons. With ongoing maturation, RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma display a different spatiotemporal regulation of mRNAs and proteins in the pre- and postnatal retina. Finally, in adulthood these six RPTPs localize to distinct cellular compartments of multiple retinal neurons. Additional studies in RPTPgamma(-/-) and RPTPbeta/zeta(-/-) (also known as PTPRZ1, RPTPbeta or RPTPzeta) mice at postnatal stage P1 reveal no apparent differences in retinal laminar organization or in the expression pattern of specific retinal cell-type markers when compared with wild type. However, in RPTPbeta/zeta(-/-) retinas, immunoreactivity of vimentin, a marker of Müller glial cells, is selectively reduced and the morphology of vimentin-immunoreactive radial processes of Müller cells is considerably disturbed. Our results suggest distinct roles of RPTPs in cell proliferation and establishing phenotypes of different retinal cells during retinogenesis as well as later in the maintenance of mature retina.

Accruing evidence indicates that gap junctions are involved in neuronal survival after brain injury. The present study was aimed at clarifying the contribution of the neuronal gap-junction protein connexin36 (Cx36) to secondary cell loss after injury in the mouse retina. A focal retinal lesion was induced by infrared laser photocoagulation. Remarkably, this model allowed spatial and temporal definition of the lesion with high reproducibility. Moreover, Cx36 is abundantly expressed in the retina and plays an essential role in the visual transmission process. Taking advantage of these features, cell death was assessed using TUNEL assay and light and electron microscopy, and the extent of Cx36 expression was studied by immunohistochemistry, Western blot, in situ hybridization and real-time RT-PCR. Secondary cell loss was most prominent between 24 and 48 h after lesioning. This peak was accompanied by an increase in Cx36 expression. When cultured explanted retinas were subjected to gap-junction blockers a significant increase in the extent of secondary cell loss after laser photocoagulation became evident. Using the same experimental paradigm we compared the incidence of cell death in wild-type and Cx36(-/-) mice. A significant increase in total number of TUNEL-positive cells occurred in the Cx36(-/-) mice compared to controls. From these data we conclude that Cx36 contributes to the survival and resistance against damage of retinal cells and thus constitutes a protective factor after traumatic injury of the retina.

Gap junction-mediated electrical coupling contributes to synchronous oscillatory activities of neurons, and considerable progress has been made in defining the molecular composition of gap junction channels. In particular, cloning and functional expression of gap junction proteins (connexins (Cx)) from zebrafish retina have shown that this part of the brain possesses a high degree of connexin diversity that may account for differential functional properties of electrical synapses. Here, we report the cloning and functional characterization of a new connexin, designated zebrafish Cx52.6 (zfCx52.6). This connexin shows little similarity to known connexins from fish and higher vertebrates. By combining in situ hybridization with Laser Capture Microdissection and RT-PCR, we found that this novel fish connexin is expressed in horizontal cells in the inner nuclear layer of the retina. Functional expression of zfCx52.6 in neuroblastoma cells and Xenopus oocytes led to functional gap junctional channels and, in single oocytes, induced large non-junctional membrane currents indicative of the formation of hemichannels, which were inhibited in reversible fashion by raising extracellular Ca(2+) concentrations.

Highlights • Zebrafish Cx52.6 is alternatively spliced in the retina. • The spliced isoform (Cx53.4) is expressed in all subtypes of horizontal cells. • Cx53.4 forms dendro-dendritic and axo-axonal gap junctions between zebrafish horizontal cells. Abstract Retinal horizontal cells (HCs) are inhibitory neurons, which modulate the transmission of light-elicited signals from photoreceptors to bipolar cells in the outer retina. HCs of the same physiological type are extensively coupled via gap junctions. In the zebrafish retina, the population of HCs comprises up to four morphologically distinct subtypes. Four different connexins (Cx52.6, Cx52.7, Cx52.9 and Cx55.5) were detected in these cells with overlapping expression patterns. In this study, we show that Cx52.6 is alternatively spliced in the retina, resulting in an additional isoform, designated as Cx53.4, which differs from the originally described Cx52.6 only by the final C-terminal peptide (12 vs. 4 aa). Further protein sequence alignments revealed that Cx53.4 represents the counterpart of alternatively spliced mouse Cx57 and human Cx62. RT-PCR analyses of mRNA expression in different adult zebrafish tissues showed that Cx53.4 is expressed exclusively in the retina. The localization of Cx53.4 protein within the retina was analyzed using a specific antibody. Immunofluorescence analyses demonstrated that the expression of Cx53.4 is restricted to HCs of all four subtypes. Further, immunoelectron microscopy confirmed the presence of Cx53.4 in gap junctions between HC dendrites and between their axon terminals. [ABSTRACT FROM AUTHOR]

Abstract: In the retina, chemical and electrical synapses couple neurons into functional networks. New candidates encoding for electrical synapse proteins have recently emerged. In the present study, we determined the localization of the candidate protein pannexin1 (zfPanx1) in the zebrafish retina and studied the functional properties of zfPanx1 exogenously expressed in Neuroblastoma 2a (N2a) cells. zfPanx1 was identified on the surface of horizontal cell dendrites invaginating deeply into the cone pedicle near the glutamate release sites of the cones, providing in vivo evidence for hemichannel formation at that location. This strategic position of zfPanx1 in the photoreceptor synapse could potentially allow modulation of cone output. Using whole cell voltage clamp and excised patch recordings of transfected N2a cells, we demonstrated that zfPanx1 forms voltage-activated hemichannels with a large unitary conductance in vitro. These channels can open at physiological membrane potentials. Functional channels were not formed following mutation of a single amino acid within a conserved protein motif recently shown to be N-glycosylated in rodent Panx1. Together, these findings indicate that zfPanx1 displays properties similar to its mammalian homologues and can potentially play an important role in functions of the outer retina. [Copyright &y& Elsevier]