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Krista Beach, Hope Queener, Takae Kiyama, Steven Wang, Deborah Otteson; Expression of the Axonal Guidance Receptors EPHA5 and EPHA6 Changes Across Retinal Development. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5147.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cell therapies to restore functional vision to patients with optic nerve disease will require new retinal ganglion cells (RGCs) to project to visual areas of the brain and form appropriate synapses. Signaling through tyrosine kinase ephrin receptors EPHA5 and EPHA6 on RGCs help pattern the retinotopic organization of RGC synapses in visual centers of the brain during development. EphA5 and EphA6 mRNAs are expressed in temporal-high/nasal-low gradients in the ganglion cell layer (GCL). The purpose of this research is to determine the spatial distribution of EPHA5 and EPHA6 proteins during development and the role of POU4F2, a transcriptional activator of EphA5 in vitro, in regulating the spatial distribution of EPHA5 and EPHA6.
Eyes from C57BL/6 (pigmented), B6(Cg)-Tyr2c-J/J (unpigmented), and Pou4f2-/--Tyr2c-J mice were collected at embryonic day 14.5 (E14.5) and post-natal day 0 (P0). Eyes were fixed, cryoprotected, and sectioned along the nasal-temporal axis. After double-immunostaining for EPHA5 and EPHA6, fluorescence images of the nasal and temporal retinal tips were captured with a monochrome CCD camera. Masks were applied to remove non-retinal areas and isolate the GCL (Adobe Photoshop). Mean pixel intensities for the full retinal thickness and for the GCL were measured (ImageJ). Background intensities of each unmasked image were subtracted. Temporal/nasal ratios were calculated and compared using T-tests.
At E14.5, robust EPHA5 immunoreactivity was present in the GCL in all strains. EPHA6 staining was similar, but less intense. At P0, EPHA5 staining was still predominant in the GCL, but EPHA6 staining increased, co-localizing with EPHA5 in the GCL. EPHA6 staining was stronger in the retinal nerve fiber layer (RNFL) vs. GCL, while EPHA5 was not in the RNFL. Qualitatively EPHA5 and EPHA6 staining intensities appeared somewhat higher in temporal vs. nasal GCL in many eyes, but differences did not reach statistical significance. There was no quantitative loss of EPHA5 or EPHA6 immunoreactivity in retinas of Pou4f2 knockout mice.
Nasal-temporal differences in EPHA5 and EPHA6 protein levels are more subtle than is known for their mRNAs. Enrichment of EPHA6 in the axons of RGCs suggests a more important role in axon pathfinding and/or connectivity than previously appreciated. POU4F2 is not required for normal EPHA5 and EPHA6 expression in retina.
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