Abstract
Purpose::
Protein diversity cannot be fully characterized by gene expression analysis alone, there is often poor correlation between mRNA expression and protein concentration; often due to events such as alternative splicing and posttranslational modifications.Protein expression analysis enables investigation of normal cell function and the changing cellular events that reflect normal developmental processes such as retinogenesis.The chick visual system is recognised as one of the most suitable tools to study molecular mechanisms of neural development, consequently proteomic profiling of the developing retina will generate information about the complex events that drive neurogenesis and latterly synaptogenesis.
Methods::
Retinae from White leghorn embryos (Gallus galllus domesticus) were extracted in lysis buffer. One milligram of protein was arrayed in the first dimension on IPG 4-7 and in the second dimension on 12% polyacrylamide gels which were visualised with Coomassie blue, densitometrically scanned and analysed using Progenesis software. Proteins of interest were excised, tryptically digested, and identified by ESI-MS.
Results::
A number of differentially expressed proteins that could be correlated with specific developmental timepoints in the embryonic retina were identified. Alpha enolase, an enzyme involved in glycolysis displayed a 26-fold increase from embryonic day (ed)7 to post hatch day 1 and eight alpha enolase isoforms were detected on 2D gels. Expression of gamma actin, a major component of the cytoskeleton, increased 3.4 fold between ed12-ed13 and >3 fold between ed17-ed19; this corresponds with formation of the outer plexiform and inner plexiform layers and is concurrent with histological evidence for the differentiation of the OPL and IPL. Fatty acid binding protein 7 (FABP7), a small cytosolic carrier of hydrophobic ligands, showed increased expression levels from ed7 - ed9 when expression was maximal, followed by a gradual decrease in expression, suggesting that FABP may play a role in the early stages of retinal differentiation.
Conclusions::
The present study is the first to commence analysis of the developing embryonic chick neural retinal proteome. Such global evaluations have begun to identify proteins that exhibit modulation during the critical phases of neurogenisis and synaptogenesis. This essential proteome data will provide insight into the poorly understood and complex events underpinning retinogenesis and perhaps offer insight into retinal degenerative disorders.
Keywords: proteomics • retinal development