Retinal Ganglion Cell-specific Transcription Factors POU4F2 and ISL2 are Positive Regulators of the Axonal-guidance Gene EphA5

Abstract

 

Purpose:

 

EPHA5 is an ephrin receptor that patterns the retinotopic maps of retinal ganglion cell (RGC) axons in the visual system. In the mouse retina, EphA5 mRNA is expressed in a temporal>nasal gradient in RGCs. However, the mechanisms regulating EphA5 expression are not well understood. This study addresses the role of ganglion cell-specific transcription factors in regulating EphA5 expression in the retina.

 

Methods:

 

Transcription factor binding sites were identified by bioinformatics using TESS and Genomatix software. Effects of overexpressing Pou4f2, Isl2, or Zic2 on the mouse EphA5 promoter activity were tested using dual luciferase assays. Expression of EPHA5 protein was assessed by immunofluorescence in histological sections from C57BL/6, B6(Cg)-Tyr^c-2J/J, and Pou4f2 null mice (P0.5).

 

Results:

 

Potential binding sites for POU4F, ISL and ZIC transcription factors were identified in the mouse EphA5 proximal promoter. In luciferase assays, deletion of regions containing POU4F and/or ISL binding sites decreased EphA5 promoter activity. In contrast, deletion of regions containing ZIC binding sites increased EphA5 promoter activity. Co-transfection of Pou4f2 or Isl2 activated and Zic2 repressed EphA5 promoter activity. In C57BL/6 (pigmented) and B6(Cg)-Tyr^c-2J/J (unpigmented) mice at P0.5, EPHA5 protein was highly expressed in the ganglion cell layer (GCL) with only subtle differences between nasal and temporal retina. In Pou4F2 null mice on a B6(Cg)-Tyr^c-2J/J background, EPHA5 immunoreactivity in the nasal retina was greatly reduced despite a large number of cells remaining in the GCL. In contrast, EPHA5 immunoreactivity in the temporal retina was robust, resulting in a clear temporal to nasal gradient.

 

Conclusions:

 

POU4F2 and ISL2 are transcriptional activators, and ZIC2 is a repressor, of the EphA5 promoter in vitro. Persistence of EPHA5 expression in Pou4F2 null retinas indicates that additional factors compensate for loss of POU4F2. The nasal/temporal differences in EPHA5 expression in Pou4F2 null mice could reflect different requirements for POU4F2 in nasal vs. temporal retina or a previously unappreciated topographic difference in RGC loss in these mice.

 

Keywords: gene/expression • transcription factors • retinal development