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Maria Blixt, Finn Hallböök; Lineage tracing of horizontal and photoreceptor cells in the embryonic chicken retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1766.
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© 2017 Association for Research in Vision and Ophthalmology.
It has previously been shown, by time-lapse studies in the zebra fish retina, that horizontal cells (HCs) and cone photoreceptor cells (PRs) share an immediate common progenitor cell. We therefore here sought to establish a method allowing us to specifically label and trace PRs and HCs in the embryonic chicken retina, thereby allowing us to perform detailed lineage studies.
We used a regulatory element from the retinoid X receptor gamma (RXRγ) gene, which is expressed in PR progenitor cells. This, in combination with Cre-LoxP recombination, the piggyBac transposon technique and in ovo electroporation, gives constitutive expression of GFP in cells that have expressed RXRγ. We injected DNA plasmids (Fig. 1A) subretinally at stage (st) 22, 25 and 28 (Embryonic day [E] 3.5, 4.5 and 5.5), electroporated and analysed the GFP expressing cells at st37-40 (E11-14) using histological methods.
Analysis of electroporated retinas revealed GFP+ cells in the outer- (ONL), and inner nuclear layers (INL), and in the ganglion cell layer (GCL; Fig. 1B). Quantification of GFP+ cells in retinas electroporated at st22 (Fig. 1C) revealed that 74±3% of the cells were localised to the ONL, 23±4% to the INL and 1±1% to the GCL (mean ± SD, >600 cells counted, n=4). Retinas electroporated at st25 and st28 had a similar distribution (Fig. 1C). Based on immunohistochemistry the GFP+ cells co-stained with markers for PRs or HCs (Fig. 1D).
We conclude that by using the RXRγ element in combination with the piggyBac integration system it is possible to label and trace the generation of HCs and PRs in the developing chicken retina. This opens up not only for lineage tracing but also for the possibility to direct expression of effector genes, such as regulators of cell cycle progression or of cell fate, to these cells.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Specific labelling of photoreceptor and horizontal cells in the embryonic chicken retina. (A) DNA plasmids used. (B) GFP expression in an E14 retina. (C) Distribution of GFP+ cells, mean ± SD. (D) Fluorescence micrographs showing GFP and Visinin/Lim1/Isl1 immunoreactivity. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, st: developmental stage.
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