The retina proteome

The retina is a multilayered neural tissue located in the eye and converts light into nerve signals that are transported to the visual centers in the brain by the optical nerve. The retina originates from neuroepithelium (an outpocketing of the diencephalon) and consists of a network of neuronal and glial cells connected via synapses. Consequently, it shares the developmental origin and organization with other parts of the central nervous system (CNS). Transcriptome analysis shows that 66% (n=13223) of all human proteins (n=20090) are expressed in the retina and 759 of these genes show an elevated expression in the retina compared to other tissue types.

  • 759 elevated genes
  • 124 enriched genes
  • 236 group enriched genes
  • Retina has most group enriched gene expression in common with brain


Figure 1. Schematic overview of the organization of the retina. Pigment epithelium cells are located nearest the choroid, while the nerve fiber layer is closest to the vitreous humor of the eye.


Retina transcriptome

Transcriptome analysis of the retina can be visualized with regard to the specificity and distribution of transcribed mRNA molecules (Figure 1). Specificity illustrates the number of genes with elevated or non-elevated expression in the retina compared to other tissues. Elevated expression includes three subcategory types of elevated expression:

  • Tissue enriched: At least four-fold higher mRNA level in retina compared to any other tissues.
  • Group enriched: At least four-fold higher average mRNA level in a group of 2-5 tissues compared to any other tissue.
  • Tissue enhanced: At least four-fold higher mRNA level in retina compared to the average level in all other tissues.

Distribution, on the other hand, visualizes how many genes have, or do not have, detectable levels (nTPM≥1) of transcribed mRNA molecules in the retina compared to other tissues. As evident in Table 1, all genes elevated in retina are categorized as:

  • Detected in single: Detected in a single tissue
  • Detected in some: Detected in more than one but less than one-third of tissues
  • Detected in many: Detected in at least a third but not all tissues
  • Detected in all: Detected in all tissues

A. Specificity

B. Distribution

Figure 2. (A) The distribution of all genes across the five categories based on transcript specificity in retina as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (nTPM≥1) in retina as well as in all other tissues.

As shown in Figure 1, 759 genes show some level of elevated expression in the retina compared to other tissues. The three categories of genes with elevated expression in retina compared to other organs are shown in Table 1. In Table 2, the 12 genes with the highest enrichment in retina are defined.

Table 1. The number of genes in the subdivided categories of elevated expression in retina.

Distribution in the 36 tissues
Detected in singleDetected in someDetected in manyDetected in all Total
Specificity
Tissue enriched 28592314 124
Group enriched 0162677 236
Tissue enhanced 314419062 399
Total 3136528083 759


Table 2. The 12 genes with the highest level of enriched expression in retina. "Tissue distribution" describes the transcript detection (nTPM≥1) in retina as well as in all other tissues. "mRNA (tissue)" shows the transcript level in retina as nTPM values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in retina and the tissue with the second-highest expression level.

Gene Description Tissue distribution mRNA (tissue) Tissue specificity score
RHO rhodopsin Detected in single 4097.5 6147
OPN1LW opsin 1, long wave sensitive Detected in single 259.0 2590
GNAT1 G protein subunit alpha transducin 1 Detected in some 1129.9 905
SAMD7 sterile alpha motif domain containing 7 Detected in single 135.3 646
GNGT1 G protein subunit gamma transducin 1 Detected in some 1918.8 645
CABP5 calcium binding protein 5 Detected in single 392.0 600
PDC phosducin Detected in some 674.3 488
SAG S-antigen visual arrestin Detected in some 1478.8 475
SMIM40 small integral membrane protein 40 Detected in single 47.1 472
PPEF2 protein phosphatase with EF-hand domain 2 Detected in single 240.8 462
OPN1MW2 opsin 1, medium wave sensitive 2 Detected in single 46.1 462
RCVRN recoverin Detected in some 467.8 338


Protein expression of genes elevated in retina

In-depth analysis of the elevated genes in retina using antibody-based protein profiling allowed us to understand the distribution of the retina specific genes and their expression profiles. Proteins expressed in specific cell types or synapses were identified among the genes with elevated expression in the retina.

Proteins specifically expressed in pigment epithelial cells

Pigment epithelial cells aid the photoreceptor cells in various ways, including the supply of molecules essential for the conversion of light into nerve signals (phototransduction), that takes place in the photoreceptor cells. Retinaldehyde binding protein 1 (RLBP1) carries the molecules 11-cis-retinaldehyde and 11-cis-retinol and mutations in this gene have been shown to lead to different conditions causing vision loss, e.g. Cone-rod dystrophy. Beta-carotene oxygenase 2 (BCO2) is an enzyme involved in the biosynthesis of vitamin A, which is essential for phototransduction. Vitamin A is known to be transported to the retina from other parts of the body via blood circulation, however, the presence of BCO2 in pigment epithelial cells may indicate an additional site of vitamin A synthesis.


RLBP1

BCO2

Proteins specifically expressed in photoreceptor cells

An example of a protein located in the nuclei of photoreceptor cells is cone-rod homeobox (CRX), a transcription factor essential for normal photoreceptor function. There are two types of photoreceptor cells: rods and cones. In rods, phototransduction begins when light activates rhodopsin (RHO), followed by interaction with the rod-specific transducin. A component of the latter is G protein subunit gamma transducin (GNGT1). Another protein expressed in the outer segments is cyclic GMP-phosphodiesterase 6G (PDE6G). It converts cyclic GMP into 5' GMP in rods, a process known to lead to hyperpolarization and subsequent nerve signaling. S-antigen visual arrestin (SAG) turns off phototransduction by preventing interaction between rhodopsin and transducin. This allows activated rods to recover and consequently adapt to low light conditions.


CRX

RHO

GNGT1


PDE6G

SAG

Proteins involved in phototransduction in cones include g protein subunit gamma transducin 2 (GNGT2), guanylate cyclase activator 1B (GUCA1B) and arrestin 3 (ARR3). GNGT2 is a component of the cone-specific transducin, and like its rod-specific relative GNGT1, it is located in the outer segments. GUCA1B is a calcium-binding protein that activates photoreceptor guanylate cyclases. ARR3 is the cone-specific equivalent of SAG, and it is involved in the deactivation of cones.


GNGT2

GUCA1B

ARR3

Proteins specifically expressed in the outer limiting membrane

The outer limiting membrane is a row of cell junctions located where the inner and outer segments of photoreceptors connect. Crumbs 2 (CRB2) is a component of the Crumbs cell polarity complex, a protein important for many cellular processes during embryonic development. Mutation in this gene has been reported to cause congenital disorders such as retinitis pigmentosa where photoreceptor cells degrade and disappear. Transmembrane protein 221 (TMEM221) is another protein expressed in the outer limiting membrane. Its function is yet to be discovered.


CRB2

TMEM221

Proteins specifically expressed in the inner nuclear layer and ganglion cells

A transcription factor that may be involved in the development of cells in the inner nuclear layer (INL) is a visual system homeobox 2 (VSX2). VSX2 is expressed in the nuclei of a subset of cells in the INL. Recoverin (RCVRN), similarly to SAG, is known to block phototransduction in rods in darkness. Here we show that RCVRN is expressed in photoreceptor cells and in a subset of cells in INL. Calcium binding protein 5 (CABP5), is a member of a protein family involved in intracellular signaling. However, the exact function of this gene is unknown. Our analysis shows that CABP5 is expressed in the INL and ganglion cells in the human retina.


VSX2

RCVRN

CABP5

Proteins specifically expressed in the inner and outer plexiform layers

The outer and inner plexiform layers (OPL and IPL) contain synapses through which the nerve signals are transmitted between the different cells in the retina. Anoctamin 2 (ANO2) encodes a calcium-activated chloride channel expressed exclusively in the OPL. CPLX4 is a protein located in the IPL and may be involved in synaptic vesicle exocytosis. Glutamate metabotropic receptor 6 (GRM6) is, as the name suggests, one of several types of metabotropic receptors that bind the neurotransmitter L-glutamate in the central nervous system. In the retina, GRM6 is detected in both the OPL and IPL.


ANO2

CPLX4

GRM6


Gene expression shared between retina and other tissues

There are 236 group enriched genes expressed in the retina. Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, including retina, compared to all other tissues.

To illustrate the relation of retina to other tissue types, a network plot was generated, displaying the number of genes with a shared expression between different tissue types (Figure 3).

Figure 3. An interactive network plot of the retina enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of retina enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to 4 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.


Retina shares most group enriched gene expression with the brain (n=180), which is expected based on that retina and brain share developmental origin and organization. GNG13 is a protein expressed exclusively in synapses in the retina and brain, as well as a subset of cells in the INL of the retina. It is a gamma subunit of a heterotrimeric G protein, which is known to be expressed in taste, retinal and neuronal tissues.


GNG13 - retina

GNG13 - cerebellum


Proteins analyzed in retina

Protein expression of selected genes with RNA expression elevated in retina or literature indicating function related to retina were analyzed in retina samples. The full list of genes used for protein profiling in the retina is found in Table 3.

Table 3. Following 112 genes have been analyzed in retina.

Gene Gene description Tissue Staining pattern
ABCA4 ATP binding cassette subfamily A member 4 Retina Staining in photoreceptor cells.
ABCG4 ATP binding cassette subfamily G member 4 Retina Staining in photoreceptor, plexiform and nerve fiber layers.
AC010319.2 Novel protein Retina Staining mainly in outer limiting membrane.
AIPL1 Aryl hydrocarbon receptor interacting protein like 1 Retina Positivity in photoreceptor cells.
ANKRD33 Ankyrin repeat domain 33 Retina Staining in photoreceptors.
ANO2 Anoctamin 2 Retina Positivity in outer plexiform layer.
ARL3 ADP ribosylation factor like GTPase 3 Retina Positivity in all layers of retina.
ARMS2 Age-related maculopathy susceptibility 2 Retina Positivity in outer and inner plexiform layers.
ARR3 Arrestin 3 Retina Staining in cones.
ATP1B2 ATPase Na+/K+ transporting subunit beta 2 Retina Staining in most layers, strongest in photoreceptor cells and inner nuclear layer.
ATP8A2 ATPase phospholipid transporting 8A2 Retina Staining in photoreceptor and ganglion cells and a subset of cells in inner nuclear layer.
BCO2 Beta-carotene oxygenase 2 Retina Staining in photoreceptor cells and pigment epithelium.
BEST1 Bestrophin 1 Retina Positivity in basal membrane of retinal pigment epithelium.
BFSP1 Beaded filament structural protein 1 Eye Moderate cytoplasmic positivity was observed in lens.
BFSP2 Beaded filament structural protein 2 Eye Strong positivity was observed in lens.
BTBD6 BTB domain containing 6 Eye Strong positivity was observed in lens.
CABP5 Calcium binding protein 5 Retina Positivity in ganglion cells and cells in inner nuclear layer.
CACNA1F Calcium voltage-gated channel subunit alpha1 F Retina Positivity in photoreceptor cells segments.
CDHR1 Cadherin related family member 1 Retina Positivity in photoreceptor cell outer segments.
CFAP161 Cilia and flagella associated protein 161 Retina Staining in cones and nerve fibers.
CHRNA3 Cholinergic receptor nicotinic alpha 3 subunit Retina Positivity in cells in photoreceptor layer, nerve fibers in outer and inner plexiform layer and nerve fiber layer.
CLIC6 Chloride intracellular channel 6 Retina Positivity in nerve fibers in nerve fiber layer.
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Retina function

The retina is a multilayered neural tissue that comprises the innermost layer of the eye, called the inner photosensitive layer. Similar to other parts of the central nervous system, it originates from the neuroepithelium, an outpocketing of the diencephalon. In the retina, light is converted into nerve signals that are transported to the visual centers in the brain by the optical nerve. As light enters the eye through the pupil and becomes refracted by the lens, it reaches the retina where it is absorbed. Photosensitive neuronal cells called photoreceptor cells transduce the light into nerve signals (this process is also known as phototransduction), which are then transmitted to different types of interneurons that modulate the nerve signaling before it is collected in the optical nerve.

There are two types of photoreceptor cells: rods and cones. Rod photoreceptors cells register the presence of light and allow vision during low light conditions while cones register red, green and blue colors and allow color vision. A layer of pigment epithelial cells provides nutrients, protection against UV radiation, and molecules essential for phototransduction to the photoreceptor cells. Four types of interneurons modulate nerve signaling by inhibiting and/or activating the cells they interact with, all located in the inner nuclear layer. Horizontal cells connect only to synapses of the photoreceptor cells, while bipolar cells transmit signals between photoreceptor cells and ganglion cells. Amacrine cells modulate signals coming from bipolar cells to ganglion cells. Ganglion cells are the last cells to receive and regulate molecular signals from the photoreceptor cells before nerve signals are collected in the optical nerve. They interact both with bipolar and amacrine cells. Muller glia cells, as the name suggests are glial cells. They maintain the structural integrity of the retina by stretching across all layers as well as buffer potassium and neurotransmitters.


Background

Here, the protein-coding genes expressed in retina are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize corresponding protein expression patterns of genes with elevated expression in retina.

Transcript profiling was based on a combination of two transcriptomics datasets (HPA and GTEx), corresponding to a total of 14590 samples from 55 different human normal tissue types. The final consensus normalized expression (nTPM) value for each tissue type was used for the classification of all genes according to the tissue-specific expression into two different categories, based on specificity or distribution.


Relevant links and publications

Uhlén M et al., Tissue-based map of the human proteome. Science (2015)
PubMed: 25613900 DOI: 10.1126/science.1260419

Yu NY et al., Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium. Nucleic Acids Res. (2015)
PubMed: 26117540 DOI: 10.1093/nar/gkv608

Fagerberg L et al., Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics. (2014)
PubMed: 24309898 DOI: 10.1074/mcp.M113.035600