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Mario Guido, Luis Morera, Nicolás Díaz; CHARACTERIZATION OF CHICKEN RETINAL HORIZONTAL CELL PRIMARY CULTURES. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2007.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cells (RGCs) expressing the photopigment melanopsin (Opn4) display intrinsic photosensitivity. In the chicken retina, two Opn4 genes, the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs have been described. Opn4x was limited to the forming ganglion cell (GC) layer and optic nerve at embryo day 8 (E8), but by E15 its expression was mostly in Prox1 (+) horizontal cells (HCs) (Verra et al., 2011). The aim of this work was to purify HCs from the chicken retina to obtain primary cultures for further characterization.
Disaggregated chicken embryonic retinas at E15 were subject to a discontinuous bovine serum albumin (BSA) gradient of 1 to 4% concentrations (Morera et al., 2012). After centrifugation, cells collected from the different phases were cultured for 4 days and characterized by immunochemistry and cell morphology. Phases were examined with specific antibodies against Opn4x, HC markers (Prox-1, Islet-1, calretinin) and markers for other retinal cell populations. Prox-1 expression was also determined by flow cytometry in cells from the whole disaggregated retina or cells from the 2.5% BSA gradient. Retinal cells from 2.5 % BSA gradient were immunopurified with an anti-OPN4x antibody and characterized by immunocytochemistry. mRNA expression was assessed by RT-PCR for retinal cell specification markers such as Brn3 and Prox-1, G proteins (Gq and α-trans) and Opn4x.
The results show that only the fraction corresponding to 2.5% BSA contained most cells displaying PROX-1 and Islet-1 positive immunoreactivities with a typical HC morphology. Based on an accurate morphological analysis, a number of cells in this fraction resembled H1- and H3-types of HCs. Strikingly, Opn4x-immunoreactivity was observed in cultures from both the 2.5 and 3 % BSA gradient phases. It is noteworthy that the 3% phase contains cells that express the neuronal filament of 200 KDa (NF200) and display longer processes that morphologically resemble RGCs. By flow cytometry we found a 30% of cells from the whole disaggregated retina and 80% of cells from the 2.5% of BSA gradient that were positively labeled for Prox-1 respectively. After immunopurification, cultures contained only OPN4x (+) HCs which were also found to express the Gq mRNA.
In conclusion, by means of this method we selectively obtained primary cultures highly enriched in HCs expressing non-visual photoreceptor components.
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