As an initial step to better understand rod photoreceptor development we profiled the proteome of the developing mouse retina during the time of maximal rod photoreceptor genesis and cell fate determination. To make the expression analysis more robust, we analyzed retinas from ages embryonic day (E)13, E15, E17 E18 P0 and P5. Representative gels from each age are shown in Figure . Expression values for each protein spot were used to cluster spots based on their changing levels of expression from E13 to P5. Figure shows the SOM clustering results when 6 clusters were pre-specified. The resulting clusters contained groups of proteins that had their peak in expression at each of the ages examined. For this analysis, we were most interested in the clusters that contained proteins that peaked at E17, which is just prior to the peak of rod photoreceptor genesis, P0 which is at the peak of rod photoreceptor genesis and P5, which is past the time of rod genesis, but the time when early, irreversible rod differentiation is occurring.
Representative images of gels from embryonic and postnatal retinal protein samples. Proteins were separated first by isoelectric focus point (pH 3-10) then by molecular weight (kDa).
Figure 2 Changes in protein expression across developmental time were used to cluster protein spots into six groups (c0-c5). Each group contained protein spots whose expression peaked at a particular developmental age. In each panel the y-axis represents relative (more ...)
Based on the clustering analysis, spots in cluster 1 (c1; expression peaked at E17), c4 (expression peaked at P0) and c0 (expression peaked at P5) were hand-picked for identification. Of the spots that were picked for analysis, 71.1% (170/239) returned high probability IDs that could be confirmed based on known or predicted molecular weights and isoelectric focus points (pIs). However, some spots returned two different identities, likely because the spots contained both proteins. These spots were not considered further. The resulting dataset, then, included 60 spots, that represented 42 unique proteins. Tables , and 3 list the protein spots whose expression peaked at E17, P1 and P5 respectively.
Dynamically expressed retinal proteins that peaked at E17.
Dynamically expressed retinal proteins that peaked at P0.
To better understand the proteins that were identified in this analysis, we did a manual literature search to look for published links between each protein and normal retinal development and brain development. Of 60 protein spots whose expression peaked at E17, 16 were identified. Based on a search of the literature, 5 proteins that peaked at E17 had been previously linked to retinal development and 3 to brain development (Table and Figure ). Of 56 protein spots whose expression peaked at P0, 7 were identified. Based on a search of the literature, 2 proteins had been previously linked to retinal development and 1 to brain development (Table and Figure ). Of 123 protein spots whose expression peaked at P5, 36 were identified. Based on a search of the literature, 12 had been previously linked to retinal development and 5 to brain development (Table and Figure ).
Proteins whose expression peaked at E17. Protein spots, on a representative 2D gel from an E17 mouse retina protein sample are labeled by spot numbers given in table 1.
Proteins whose expression peaked at P0. Protein spots, on a representative 2D gel from a P0 mouse retina protein sample are labeled by spot numbers given in table 2.
Dynamically expressed retinal proteins that peaked at P5.
Proteins whose expression peaked at P5. Protein spots, on a representative 2D gel from a P5 mouse retina protein sample are labeled by spot numbers given in table 3.
This analysis identified 42 distinct proteins that are dynamically expressed in the retina during rod photoreceptor development. Of these proteins, 10 were represented by more than one protein spot, suggesting they are dynamically post-translationally modified. Finally, a manual search of the published literature identified prior published reports had already linked 16 of the 42 proteins to retinal development in some way.
The proteins reported here most certainly do not constitute a complete list of molecules dynamically expressed during development. A number of proteins already demonstrated to be important during photoreceptor development do not appear in our dataset. This could be due to a number of factors including the relative abundance of a protein in the samples, relative change in it's expression levels, high-confidence identification of the protein with MALDI MS/MS, verification of the protein spot ID based on 2D gel position and the protein spot containing a single protein. Thus, while this study reports important results on it's own, we also consider it complimentary to other reports of gene or protein expression in the developing mouse retina.
A number of important studies have used expression analysis to identify genes or proteins expressed in the developing mouse retina [3
]. The motivation behind this approach is two-fold. Firstly, molecules important for particular events during retinal development may be expected to change at the time that said event is occurring. Secondly, profiling genes that change in relation to one another may help investigators to identify pathways or groups of genes that work together during retinal development. Protein expression profiling can be a powerful compliment to mRNA expression analysis. Changes in protein expression are a more definitive measure of how much gene product is present in cells. However, the most powerful compliment that 2D gel expression analysis offers is the ability to capture not only changes in expression but also changes in post-translational modification. The existence of post-translational modifications can be discovered by differences in pI or molecular weight. In our analysis alone, we identified 10 proteins likely with dynamic post-translational modifications. In future experiments specific dyes for phosphorylation and glycosylation may be useful to identify and quantify specific post-translational modifications.
A previously published complementary study used 2D-gel electrophoresis to profile dynamic changes in protein expression in the postnatal mouse retina [8
]. In this study they identified 174 total protein spots. Of the 170 total protein spots that returned identities in the current analysis (E17, P0 and P5), 47 of them were in common with the previous study. Protein expression profiling has also been successfully applied in the developing chick retina [9
]. Even though these studies may have profiled different ages and/or species it still may be useful to integrate the information from these and other studies to generate a more comprehensive profile of changes in protein expression during vertebrate retinal development.
We have used protein expression profiling to identify retinal proteins with dynamic changes in expression during rod photoreceptor genesis. We identified 16 proteins that have been previously associated with the developing retina and 26 that have not been previously associated with retinal development.