Abstract
Purpose :
Investigate the functional significance of the discrete distribution of S100A4 in lens fibers, using whole transcriptome analysis to probe differential gene expression profiles of S100A4 null and littermate wildtype mouse lenses.
Methods :
Poly(A) mRNA capture and construction of stranded mRNA-seq libraries using total RNA derived from one month-old transparent S100A4 null and littermate wild type mouse lenses, was performed using the KAPA Stranded mRNA-Seq Kit. An Illumina HiSeq 4000 flow cell was used to sequence the stranded mRNA-seq libraries at 50bp single reads. Sequences were demultiplexed using Illumina bcl2fastq conversion software version 2. Selected differentially expressed genes were confirmed by qRT-PCR, immunoblotting and immunofluorescence analyses. Changes in the levels of cyclic GMP in S100A4 and wild type mouse lenses were determined using a cGMP XPTM assay kit.
Results :
Based on analysis of two independent sample sets, global RNAseq differential gene expression profile data from mouse lenses revealed that S100A4 deficiency leads to a robust and discrete upregulation (~250-fold increase) of the gene encoding neuronal specific protein S100A5, which is expressed at negligible levels in the wild type lens. Intriguingly, S100A4 deficiency in the mouse lens was also found to be associated with significant induction of genes encoding retinal specific transcriptional factors (including Vsx2, Nrl, Crx, NR2E3, Otx and others), visual phototransduction proteins from both rods and cones, (including rhodopsin, peripherin-2, cGMP phosphodiesterases, transducin alpha-subunit, recoverin, retinal outer segment membrane protein1, rhodopsin kinase, regulator of G-protein signaling 9, cyclic nucleotide gated channel alpha 1 and others) and various neurotransmitter transporters (e.g. slc6a6) in the mouse lens. qRT-PCR, immunoblotting and immunofluorescence analyses further confirmed the expression and distribution of S100A5, rhodopsin, PDE6γ, transducin 1, CNGα1 and GRK1 in S100A4 null mouse lenses compared to wildtype lenses. cGMP levels were modestly increased in dark adapted lenses from P30 day-old S100A4 null mice compared to wildtype mice.
Conclusions :
This study uncovers the importance of S100A4 in suppressing expression of the retinal specific genes in the ocular lens, suggesting a key transcriptional repressor role for S100A4.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.