Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development

For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1–like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.

Expression and modulation of connexin30.2, a novel gap junction protein in the mouse retina

Mammalian retinae express multiple connexins that mediate the metabolic and electrical coupling of various cell types. In retinal neurons, only connexin36, connexin45, connexin50, and connexin57 have been described so far. Here, we present an analysis of a novel retinal connexin, connexin30.2 (Cx30.2), and its regulation in the mouse retina. To analyze the expression of Cx30.2, we used a transgenic mouse line in which the coding region of Cx30.2 was replaced by lacZ reporter DNA. We detected the lacZ signal in the nuclei of neurons located in the inner nuclear layer and the ganglion cell layer (GCL). In this study, we focused on the GCL and characterized the morphology of the Cx30.2-expressing cells. Using immunocytochemistry and intracellular dye injections, we found six different types of Cx30.2-expressing ganglion cells: one type of ON-OFF, three types of OFF, and two types of ON ganglion cells; among the latter was the RGA1 type. We show that RGA1 cells were heterologously coupled to numerous displaced amacrine cells. Our results suggest that these gap junction channels may be heterotypic, involving Cx30.2 and a connexin yet unidentified in the mouse retina. Gap junction coupling can be modulated by protein kinases, a process that plays a major role in retinal adaptation. Therefore, we studied the protein kinase–induced modulation of coupling between RGA1 and displaced amacrine cells. Our data provide evidence that coupling of RGA1 cells to displaced amacrine cells is mediated by Cx30.2 and that the extent of this coupling is modulated by protein kinase C.

A Battery of Cell- and Structure-specific Markers for the Adult Porcine Retina

The pig is becoming an increasingly used non-primate model in experimental studies of human retinal diseases and disorders. The anatomy, size, and vasculature of the porcine eye and retina closely resemble their human counterparts, which allows for application of standard instrumentation and diagnostics used in the clinic. Despite many reports that demonstrate immunohistochemistry as a useful method for exploring neuropathologi-cal changes in the mammalian central nervous system, including the pig, the porcine retina has been sparsely described. Hence, to facilitate further immunohistochemical analysis of the porcine retina, we report on the successful use of a battery of antibodies for staining of paraformaldehyde-fixed cryosectioned retina. The following antibodies were evaluated for neuronal cells and structures: recoverin (cones and rods), Rho4D2 (rods), transducin-γ (cones), ROM-1 (photoreceptor outer segments), calbindin (horizontal cells), PKC-α (bipolar cells), parvalbumin (amacrine and displaced amacrine cells), and NeuN (ganglion cells and displaced amacrines). For detecting synaptic connections in fiber layers, we used an antibody against synaptobrevin. For detecting retinal pigment epithelium, we studied antibodies against cytokeratin and RPE65, respectively. The glial cell markers used were bFGF (Müller cells and displaced amacrine cells), GFAP (Müller cells and astrocytes), and vimentin (Müller cells). Each staining effect was evaluated with regard to its specificity, sensitivity, and repro-ducibility in the identification of individual cells, specific cell structures, and fiber layers, respectively. The markers parvalbumin and ROM-1 were tested here for the first time for the porcine retina. All antibodies tested resulted in specific staining of high quality. In conclusion, all immunohistochemical protocols presented here will be applicable in fixed, cryosectioned pig retina.