Abstract
Purpose :
Forward and reverse signaling through ephrin ligands and their receptors (EPH) regulates formation of topographical maps in the visual system. EphA5 is expressed in a temporal high/nasal low gradient across the developing and mature retina. To identify novel regulators of the EphA5 promoter, whole transcriptome sequencing was used to analyze differential gene expression in the temporal and nasal retinal ganglion cell layer (GCL).
Methods :
Temporal and nasal thirds of the GCL from postnatal day 2 (P2) C57Bl/6J mice were isolated using laser capture microdissection for RNA sequencing (Illumina). RT-qPCR of independent RNA samples isolated from temporal (n=3) and nasal (n=3) retinal thirds used hydrolysis probes (TaqMan) and Relative Expression Software Tool analysis. Tbx5 (T-box5) subcloned into pcDNA3.1 or empty pcDNA3.1 plasmids were co-transfected with mEphA5-pGL2 luciferase promoter constructs into 293FT cells. Relative luciferase, normalized to co-transfected pCMV-βgal, was compared by ANOVA and post-hoc T-tests. Developmental expression of TBX5 in mouse retinas used immuno-fluorescence labeling and confocal microscopy. Chromatin immunoprecipitation (ChIP) used sheared chromatin from P7 retinas and antibodies against TBX5 or IgG followed by qPCR analysis .
Results :
At P2, 40 genes showed a 2-fold or greater difference in expression between temporal and nasal GCL by RNA-seq, with Tbx5 increased 5.3-fold in the temporal GCL (p=0.017 vs. nasal). By RT-qPCR, Tbx5 expression was 15.6-fold higher in temporal vs. nasal retinal RNA at P2 (p=0.001). The mouse EphA5 proximal promoter region contained four putative TBX binding sites (T-box), located between -1080 and -146. Overexpression of Tbx5 increased activity of EphA5 promoter constructs (ANOVA, p<0.001), with effect sizes ranging from 7.4-fold (p=0.04; 1 TBX site), to 18.3-fold (p=0.02; 4 TBX sites). TBX5 occupancy of the proximal EphA5 promoter in vivo was confirmed by ChIP, with a 330-fold enrichment (TBX5 vs. IgG) by qPCR. A distal T-box site, located at -2910 from the EphA5 transcription start site, was not amplified from TBX5 ChIP’ed chromatin. By immunofluorescence, TBX5 was detected in retinal ganglion cells at embryonic day14.5.
Conclusions :
During early retinal development, Tbx5 is known to play a role in dorsal/ventral patterning. Our data point to a novel role for Tbx5 in regulating nasal/temporal gene expression of EphA5 in later stages of retinal development.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.