Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Figure 51.10 Spatial regulation of gene expression in the retina using cell type–specific promoters. Retinal cell type–specific promoters were fused to DsRed cDNA and electroporated into P0 rat retinas. The retinas were harvested at P20 (A–F and H) or P2 (G),

sectioned, and stained with DAPI. Promoters of rhodopsin (A), Nrl (B), Cabp5 (C ), Ndrg4 (D), Cralbp (E), clusterin (F), and Hes1 (G and H) were used to express DsRed. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. See color plate 58.

Figure 51.11 Multicolor labeling of the retina using cell type– specific promoters. P0 rat retina was electroporated with three reporter constructs: rhodopsin promoter 2.2K-CFP (specific for rods), Cabp5 promoter 4.7K-YFP (specific for a subset of bipolar cells), and Cralbp promoter 4.0K-DsRed (specific for Müller glia). The retina was harvested at P20, sectioned, and stained with DAPI. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment. See color plate 59.

of DsRed in amacrines as well as in a subset of rods. The Cralbp promoter-Cre construct and the clusterin promoterCre construct induced the expression of DsRed in Müller glia and a small population of rod and bipolar cells. The Hes1 promoter-Cre construct and the Rax promoter-Cre construct induced the expression of DsRed in rod, bipolar, amacrine, and Müller glial cells. The expression patterns of DsRed were analyzed at P20, with the results summarized in table 51.1.

Temporal and Cell Type–Specific Regulation By combining the temporal regulation afforded by ERT2- CreERT2 with the cell type–specific regulation provided by the promoters, we could inducibly express a reporter gene specifically in a desired cell type, such as “differentiated” Müller glia. As shown in figure 51.13, the clusterin promoter was cloned upstream of ERT2CreERT2, and the resulting plasmid was coelectroporated with CALNLDsRed (recombination indicator) and CAG-GFP (transfection control) into P0 rat retinas. The retinas were stimulated with 4-OHT at P14, the time point when retinogenesis is complete (Young, 1985; Rapaport et al., 2004), and then analyzed at P16. Without 4-OHT stimulation, DsRed expression was not detected. When 4-OHT was injected into the rats, clear DsRed expression was detected in a subset of GFP-positive cells. The retinal sections showed that all the DsRed-positive cells examined were morphologically Müller glia. These results show that ERT2CreERT2 can be used with retinal cell type–specific promoters to achieve precise temporal and cell type–specific regulation in the retina.

Conditional RNAi To conditionally knock down gene expression in the retina, Cre-dependent inducible RNAi vectors utilizing the micro-RNA30 (mir30)-based shRNA expression system (Zeng et al., 2002; Paddison et al., 2004) were made. The mir30-based shRNA expression system has several advantages over conventional RNAi vectors expressing shRNAs directly from pol III promoters, such as the U6 promoter. First, shRNA can be expressed using pol II promoters (e.g., CAG promoter). Second, it is technically simple to put the regulatory elements (e.g., transcriptional stop cassette) into the expression vectors. Figure 51.14 shows the mir30-based vectors expressing shRNA under the control of the CAG promoter (CAG-mir30 and CALSL-mir30). CALSL-mir30 is a Cre-dependent inducible RNAi vector carrying a floxed transcriptional stop cassette immediately after the CAG promoter. CALSL-mir30 also was used with the rhodopsin promoter-Cre construct in the retina. P0 rat retinas were coelectroporated with four plasmids, CALSLmir30(GFPshRNA) expressing an shRNA against GFP (GFPshRNA), CAG-GFP, CAG-DsRed, and the rhodopsin promoter-Cre, and analyzed at P20. Without the Cre construct, transfected retinal cells were clearly labeled by both GFP and DsRed. With the rhodopsin promoter-Cre construct, GFP expression in the ONL was specifically silenced while that in the INL cells was not affected, demonstrating that CALSL-mir30 can be used to conditionally knock down gene expression in the retina. These experiments showed there is a very high cotransfection efficiency. In order for cells to be both red and green, they must have been successfully electroporated with both CAGGFP and CAG-DsRed. The coexpression rate of these two genes was nearly 100%, both in this case, where four plasmids were coelectroporated, and in many previous cases with two or three coelectroporated plasmids (Matsuda and Cepko, 2004).

The Cre/loxP recombination-dependent inducible vectors with the CAG promoter have several useful features. First, it is possible to inducibly express genes in specific cell types at specific time points. Second, after Cre/loxPmediated recombination, strong “output signals” driven by the CAG promoter can be obtained. Expression of fluorescent reporter genes directly from cell type–specific promoters frequently results in such low levels of expression as to render the constructs unusable for some applications, such as the detection of live-labeled cells. We found there was a much greater sensitivity for the detection of expression when cell type–specific promoters were used with Cre plus CALNL-DsRed. Finally, these vectors can be used to trace the fate of progenitor and precursor cells labeled upon Cre/loxP-mediated recombination. Using these vectors, we showed that several cell type–specific promoters are weakly active in progenitors and/or other cell types. Two explanations for this observation, which are not mutually

Figure 51.12 Lineage tracing experiments in the retina using the Cre/loxP system and cell type–specific promoters. P0 rat retinas were coelectroporated with retinal cell type–specific promoter Cre and CALNL-DsRed (recombination indicator). DsRed expression, induced by Cre/loxP-mediated recombination, was driven by the ubiquitous CAG promoter. The retinas were harvested at P20,

exclusive, can be considered. Lineage analyses using retroviral vectors have shown that clones with bipolar, Müller glia, or amacrine cells almost always also contain rods, even if the clone is of only two cells (Turner and Cepko, 1987). If these promoters are weakly active in progenitors that give rise to multiple cell types, then one might see labeling of rods, along with labeling of the cell type that normally is the only cell type to express a particular promoter. This idea is supported by two previous observations. Most of the known Müller glia–specific genes of the adult retina are also expressed in progenitors (Blackshaw et al., 2004), and two genes thought to be restricted to amacrine and horizontal cells could be observed in progenitors (Alexiades and Cepko, 1997). An additional explanation is that these promoters, transiently introduced

sectioned, and stained with DAPI. Promoters of rhodopsin (A), Nrl (B), Cabp5 (C ), Ndrg4 (D), Cralbp (E), clusterin (F), Hes1 (G), and Rax (H) were used to express Cre. Yellow arrowheads indicate the labeled rods. Blue arrowhead indicates the labeled bipolar cell. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. See color plate 60.

into the retina by electroporation, are slightly “leaky,” and such leakiness was detected by the more sensitive reporter (Cre plus CALNL-DsRed).

Future applications

There are many obvious applications for electroporation in studies of the visual system. Standard gainand loss-of- function protocols are being employed in which a gene is overor misexpressed or is targeted with RNAi. However, the flexibility offered by the high efficiency of coelectroporation enables many more types of experiments to be performed. For example, the best control for the specificity of an RNAi event is gene rescue; that is, one supplies a version of the endogenous gene that is targeted by RNAi but that

Figure 51.13 Inducible expression in the differentiated Müller glia (A). Clusterin promoter was fused to ERT2CreERT2 cDNA and coelectroporated into P0 rat retinas with CALNL-DsRed (recombination indicator) and CAG-GFP (transfection control). Retinas were stimulated with or without 4-OHT at P14 by IP injection, then harvested at P16. B, Whole-mount preparation of the trans-

does not encode the targeted sequence. This is easily done by coelectroporating the plasmid encoding the RNAi and the resistant version of the targeted gene. One can also perform epistasis experiments. For example, one can target a transcription factor by RNAi and simultaneously supply a putative target of that transcription factor whose expression is controlled by a noncognate promoter to look for rescue. One can use the recombinases to turn on expression of either the RNAi or the rescue cassette to perform rescue or knockdown in specific cells or at specific times. Promoter constructs also allow one to mark subsets of cells and follow their migration, and so on. Their physiology can be controlled by the controlled expression of genes that might silence their neuronal activity.

Another application of interest to neuroscientists is to track the synaptic partners of an electroporated cell. Since

fected retina harvested at P16 without 4-OHT stimulation. No DsRed expression was detected. C, Whole-mount preparation of the transfected retina stimulated with 4-OHT at P14 and harvested at P16. D, The retina shown in C was sectioned and stained with DAPI. Only Müller glial cells are labeled with DsRed. See color plate 61.

the transient expression following electroporation can be relatively high level, one can provide enough of a transsynaptic tracer (e.g., WGA) to find the synaptically connected partners of an electroporated cell, which itself can be readily identified by labeling with an electroporation reporter, for example, GFP. In addition, electroporation of Cre plasmids, RNAi plasmids, and the like into mice in which engineering of the germ line has been carried out is being done. For example, rather than create a mouse strain that expresses Cre under control of a specific promoter, one can simply electroporate a Cre plasmid into a mouse that has a germ line with a floxed allele of a gene of interest. Many additional applications will be discovered through the creative use of these protocols and reagents as more neuroscientists discover the ease and flexibility of this system of gene transfer.

Figure 51.14 Inducible RNAi in the retina. A, CAG-mir30: The mir30 expression cassette is expressed under the control of the CAG promoter. The mir30 expression cassette has the hairpin stem composed of siRNA sense and antisense strands (22nt each), a loop derived from human mir30 (19nt), and 125nt mir30 flanking sequences on both sides of the hairpin. The mir30 primary transcript is processed to generate the mature shRNA. CALSLmir30: Cre-dependent inducible shRNA expression vector carrying a floxed transcriptional stop cassette (3xpolyA signal sequences).

REFERENCES

Akaneya, Y., Jiang, B., and Tsumoto, T. (2005). RNAi-induced gene silencing by local electroporation in targeting brain region.

J. Neurophysiol. 93:594–602.

Alexiades, M. R., and Cepko, C. L. (1997). Subsets of retinal progenitors display temporally regulated and distinct biases in the fates of their progeny. Development 124:1119–1131.

Ali, R. R., Reichel, M. B., Thrasher, A. J., Levinsky, R. J., Kinnon, C., Kanuga, N., Hunt, D. M., and Bhattacharya, S. S. (1996). Gene transfer into the mouse retina mediated by an adeno-associated viral vector. Hum. Mol. Genet. 5:591–594.

Bennett, J., Wilson, J., Sun, D., Forbes, B., and Maguire, A. (1994). Adenovirus vector-mediated in vivo gene transfer into adult murine retina. Invest. Ophthalmol. Vis. Sci. 35:2535– 2542.

Only in the presence of Cre, the mir30 expression cassette is expressed under the control of the CAG promoter. B and C, Conditional GFP knockdown in the retina. CAG-GFP and CAGDsRed and CALSL-mir30(GFPshRNA) expressing an shRNA against GFP were coelectroporated without (B) or with (C ) the rhodopsin promoter Cre into P0 rat retinas. The retinas were harvested at P20, sectioned, and stained with DAPI. Rod-specific GFP knockdown was observed in the presence of the rhodopsin promoter Cre. See color plate 62.

Blackshaw, S., Harpavat, S., Trimarchi, J., Cai, L., Huang, H., Kuo, W. P., Weber, G., Lee, K., Fraioli, R. E., et al. (2004). Genomic analysis of mouse retinal development. PLoS Biol. 2: E247.

Branda, C. S., and Dymecki, S. M. (2004). Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6:7–28.

Casanova, E., Fehsenfeld, S., Lemberger, T., Shimshek, D. R., Sprengel, R., and Mantamadiotis, T. (2002). ER-based double iCre fusion protein allows partial recombination in forebrain. Genesis 34:208–214.

Chen, S., Wang, Q. L., Nie, Z., Sun, H., Lennon, G., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Zack, D. J. (1997). Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. Neuron 19: 1017–1030.

Cherry, T. J., and Cepko, C. L. (2008). Unpublished results. Dezawa, M., Takano, M., Negishi, H., Mo, X., Oshitari, T., and

Sawada, H. (2002). Gene transfer into retinal ganglion cells by in vivo electroporation: A new approach. Micron 33:1–6.

Feil, R., Wagner, J., Metzger, D., and Chambon, P. (1997). Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237:752–757.

Fukuchi-Shimogori, T., and Grove, E. A. (2001). Neocortex patterning by the secreted signaling molecule FGF8. Science 294:1071–1074.

Furukawa, T., Kozak, C. A., and Cepko, C. L. (1997a). Rax, a novel paired-type homeobox gene, shows expression in the anterior neural fold and developing retina. Proc. Natl. Acad. Sci. U.S.A. 94:3088–3093.

Furukawa, T., Morrow, E. M., and Cepko, C. L. (1997b). Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell 91: 531–541.

Haeseleer, F., Sokal, I., Verlinde, C. L., Erdjument-Bromage, H., Tempst, P., Pronin, A. N., Benovic, J. L., Fariss, R. N., and Palczewski, K. (2000). Five members of a novel Ca2+-binding protein (CABP) subfamily with similarity to calmodulin. J. Biol. Chem. 275:1247–1260.

Huberman, A. D., Murray, K. D., Warland, D. K., Feldheim, D. A., and Chapman, B. (2005). Ephrin-As mediate targeting of eye-specific projections to the lateral geniculate nucleus. Nat. Neurosci. 8:1013–1021.

Jomary, C., Piper, T. A., Dickson, G., Couture, L. A., Smith, A. E., Neal, M. J., and Jones, S. E. (1994). Adenovirus-mediated gene transfer to murine retinal cells in vitro and in vivo. FEBS Lett. 347:117–122.

Kachi, S., Oshima, Y., Esumi, N., Kachi, M., Rogers, B., Zack, D. J., and Campochiaro, P. A. (2005). Nonviral ocular gene transfer. Gene Ther. 12:843–851.

Kanegae, Y., Lee, G., Sato, Y., Tanaka, M., Nakai, M., Sakaki, T., Sugano, S., and Saito, I. (1995). Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res. 23:3816– 3821.

Kennedy, B. N., Huang, J., Saari, J. C., and Crabb, J. W. (1998). Molecular characterization of the mouse gene encoding cellular retinaldehyde-binding protein. Mol. Vis. 4:14–21.

Li, T., Adamian, M., Roof, D. J., Berson, E. L., Dryja, T. P., Roessler, B. J., and Davidson, B. L. (1994). In vivo transfer of a reporter gene to the retina mediated by an adenoviral vector.

Invest. Ophthalmol. Vis. Sci. 35:2543–2549.

Mathers, P. H., Grinberg, A., Mahon, K. A., and Jamrich, M. (1997). The Rx homeobox gene is essential for vertebrate eye development. Nature 387:603–607.

Matsuda, T., and Cepko, C. L. (2004). Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. U.S.A. 101:16–22.

Matsuda, T., and Cepko, C. L. (2007). Controlled expression of transgenes introduced by in vivo electroporation. Proc. Natl. Acad. Sci. U.S.A. 104:1027–1032.

Miyoshi, H., Takahashi, M., Gage, F. H., and Verma, I. M. (1997). Stable and efficient gene transfer into the retina using an HIVbased lentiviral vector. Proc. Natl. Acad. Sci. U.S.A. 94:10319– 10323.

Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199.

Paddison, P. J., Cleary, M., Silva, J. M., Chang, K., Sheth, N., Sachidanandam, R., and Hannon, G. J. (2004). Cloning of short hairpin RNAs for gene knockdown in mammalian cells. Nat. Methods 1:163–167.

Picard, D. (1994). Regulation of protein function through expression of chimaeric proteins. Curr. Opin. Biotechnol. 5:511–515.

Price, J., Turner, D., and Cepko, C. (1987). Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 84:156–160.

Punzo, C. and Cepko, C. L. (2008). Unpublished results. Rapaport, D. H., Wong, L. L., Wood, E. D., Yasumura, D., and

LaVail, M. M. (2004). Timing and topography of cell genesis in the rat retina. J. Comp. Neurol. 21:304–324.

Saito, T., and Nakatsuji, N. (2001). Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240:237–246.

Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., and Grove, E. A. (2004). Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex. Development 131:5639–5647.

Swaroop, A., Xu, J. Z., Pawar, H., Jackson, A., Skolnick, C., and Agarwal, N. (1992). A conserved retina-specific gene encodes a basic motif/leucine zipper domain. Proc. Natl. Acad. Sci. U.S.A. 89:266–270.

Tabata, H., and Nakajima, K. (2001). Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex. Neuroscience 103:865–872.

Tomita, K., Ishibashi, M., Nakahara, K., Ang, S. L., Nakanishi, S., Guillemot, F., and Kageyama, R. (1996). Mammalian hairy and Enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis. Neuron 16:723–734.

Turner, D. L., and Cepko, C. L. (1987). A common progenitor for neurons and glia persists in rat retina late in development. Nature 28:131–136.

Turner, D. L., Snyder, E. Y., and Cepko, C. L. (1990). Lineageindependent determination of cell type in the embryonic mouse retina. Neuron 4:833–845.

Young, R. W. (1985). Cell differentiation in the retina of the mouse. Anat. Rec. 212:199–205.

Zack, D. J., Bennett, J., Wang, Y., Davenport, C., Klaunberg, B., Gearhart, J., and Nathans, J. (1991). Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas. Neuron 6:187–199.

Zeng, Y., Wagner, E. J., and Cullen, B. R. (2002). Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9: 1327–1333.

The study of mouse genetics has had a tremendous impact on medical research over the last few decades. Ophthalmology is no exception and has benefited from the mouse, which exhibits significant homology with the human genome (Waterston et al., 2002). In fact, the first scientific publication of an inheritable defect in the central nervous system (CNS) in mice was rodless (subsequently shown to be a mutation in the β subunit of cGMP phosphodiesterase). Mutations in the human homologue result in retinitis pigmentosa and blindness. More recently, knockout strains have been used to predict drug actions in humans (Zambrowicz et al., 2003). Phenotypic screens are used to examine mouse knockouts for physiological parameters that are beneficial in the clinic. This chapter provides a general introduction to the importance of mouse genetics in understanding ocular development and the physiology of particular cell types and highlights several examples of significant contributions by mouse models to the discovery and development of new therapies in human ophthalmic disease.

Cell types in the retina

The neural retina and the retinal pigmented epithelium (RPE) are derived from neuroectodermal precursors in the optic vesicle, which emerges from the ventral diencephalon (Mann, 1964). The vesicle invaginates, forming the optic cup, in which the inner layer becomes the neural retina and the outer layer becomes the RPE. The neural retina in mice is organized into three cellular layers separated by plexiform layers that contain synaptic connections among retinal neurons. The laminar organization of the mouse retina was described in a monograph entitled The Structure of the Retina, first published in 1892 by the legendary anatomist, Santiago Ramón y Cajal (1972). Relying primarily on the Golgi method to label cellular elements in detail, Ramón y Cajal described the major cell types of the mouse retina. This description of retinal cytoarchitecture established the foundation for future studies aimed at understanding anatomical and physiological relationships in the vertebrate retina.

In routine histological sections, the mouse retina appears relatively simple and neatly organized into three cellular layers (figure 52.1A). Cells within these layers can be grouped

into neuronal or glial cell types. Within a given general cell type, there are often multiple morphologies and neurochemical signatures that expand the list to 50–60 different cell types (Haverkamp and Wässle, 2000). The ganglion cell layer (GCL) in mice contains one or two rows of cells, depending on the central or peripheral location of the field in view (figure 52.1B). The GCL is populated by ganglion cells, the sole projection neurons of the retina that transmit signals through the optic nerve to the central visual nuclei. The GCL also contains retinal astrocytes, displaced amacrine cells, and cells that make up the superficial retinal vasculature, endothelial cells and mural cells. The inner nuclear layer (INL) is located in the neural retina and is five to seven cell layers thick, depending on the retinal locale in view (see figure 52.1A). The INL is home to a diverse population of retinal cells, such as amacrine cells, Müller glial cells, bipolar neurons, and horizontal cells (figure 52.1B–D).

The outer nuclear layer (ONL) contains rod and cone photoreceptors that detect illumination gradients (see figure 52.1A). Approximately 10–15 rows of nuclei populate the ONL, with rods far outnumbering cones in this nocturnal species (Carter-Dawson and LaVail, 1979). With few exceptions, cone cell nuclei are located at the periphery of the ONL (figure 52.2A). Photoreceptors are further specialized into outer and inner segments. These specializations are easily observed in routine sections. Inner segments enable the synthesis and transport of proteins destined for the outer segments. The outer segments contain stacked discs of membranes that capture photons and convert this energy to chemical messengers that travel back toward the INL to ultimately affect synaptic transmission of RGCs.

Cones account for approximately 2%–3% of the photoreceptor population in mice (Carter-Dawson and LaVail, 1979; Jeon et al., 1998). Figure 52.2B shows a reliable histological marker for cone photoreceptors, the lectin peanut agglutinin (PNA), which labels the interphotoreceptor matrix associated with cone but not rod photoreceptors ( Johnson et al., 1986). Although mice lack a central specialization observed in other mammals, the density of cones (approximately 3,500 cones/mm2) is comparable to the approximately 3,000 cones/mm2 observed in the parafoveal primate retina (Curcio et al., 1990; Jeon et al., 1998). Rod

Figure 52.1 Anatomy and cell types in the adult mouse retina. A, The neural retina contains three cellular layers known as the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). Two synaptic layers connect cells in the nuclear layers, the inner plexiform layer (IPL) and the outer plexiform layer (OPL). The retinal pigmented epithelium (RPE) contains melanin and protects and supports the photoreceptors in the ONL. The choriocapillaris (CC) is also pigmented and provides vascular supply to the RPE. The sclera (Sc) supports the outer eye. B, Higher magnification view of the inner retina. Ganglion cells (gc) are located in the GCL. Amacrine cells (ac) reside in the inner

Figure 52.2 Anatomy of the outer retina. A, Phototransduction occurs in photoreceptors located in the outer nuclear layer (ONL). Cone photoreceptors are easily distinguished from the much more numerous rod photoreceptors by their heterochromatin (arrow) and location within the ONL, which is close to the inner segments (is). Outer segments (os) contain the light-sensitive pigments that capture photons. Their tips are engulfed by apical microvilli (mv)

portion of the INL and stain much less intensely than the neighboring Müller glia (mg), located in the middle region of the INL. Bipolar cells (bc) are located on the outer portion of the INL (asterisks indicate capillaries). C, Many immunohistochemical markers can be used to identify individual cell types in the mouse retina. Anti-PKCα antibodies recognize the rod bipolar cell bodies (arrows) and their synaptic terminals (asterisks) in the IPL, near the GCL. D, Anticalbindin recognizes horizontal cells (arrows) in the outer portion of the INL. For a detailed list of molecular reagents to study individual cell types in the mouse retina, the reader should consult Haverkamp and Wässle (2000).

that extend from the retinal pigmented epithelium (RPE). The section in this image was obtained from an albino mouse that lacked pigmentation in the RPE or choriocapillaris (CC). B, The PNA-lectin conjugated to FITC is an excellent marker to visualize cone outer segments and inner segments in the mouse retina. C, Rods can be visualized with antirhodopsin antibodies.

photoreceptor density averages 437,000 cells/mm2 and therefore represents approximately 97% of total photoreceptors. Rods can be identified with a variety of antibodies; antirhodopsin is one of the most commonly used (figure 52.2C ).

The first synaptic relay in light sensation occurs at the level of the outer plexiform layer (OPL) and involves the transmission between rod photoreceptors and rod bipolar cells during low light or nocturnal conditions, and between cones and cone bipolar cells under photopic or bright-light conditions. Rod bipolar cells are routinely visualized with anti-PKCα antibodies (see figure 52.1C ). These synaptic connections mark the interface between the ONL and the INL. Unlike other neuronal cell types in the INL, horizontal cells are defined by a single morphology, the axon-bearing horizontal cell. Calbindin immunoreactivity (see figure 52.1D) is an excellent marker for horizontal cells in the mouse retina (Haverkamp and Wässle, 2000). Rod photoreceptors establish contact at the axon terminal system and cone photoreceptors provide synaptic input to the horizontal cell dendrite. Horizontal cells provide reciprocal communication to photoreceptors and input to bipolar cells. Cone and rod bipolar cells relay visual stimuli from photoreceptors to a synaptic-rich region known as the inner plexiform layer (IPL), which contains connections formed among bipolar, amacrine, and ganglion cells (see figure 52.1A and B).

The retinal pigmented epithelium

Photoreceptors are intimately associated with an adjacent layer of cells in the outer retina known as the retinal pigmented epithelium. This epithelium is derived from neuroectoderm that also differentiates into the neural retina (Strauss, 2005). Thus, both neural retina and RPE are lateral extensions of the CNS. RPE cells appear cuboidal in cross section and hexagonal when viewed in whole-mount preparations. The RPE is one of the most intriguing cell types in the eye because of its location and functions within the retina proper. RPE cells are highly polarized, exhibiting a basal side that faces Bruch’s membrane and an apical side that extends microvilli to surround the adjacent photoreceptor outer segments (see figure 52.2A). As the name implies, this epithelium contains numerous melanin granules, which give the cell a dark brown appearance in routine histological preparations (see figure 52.1A). The RPE is the first cell type in the body to express pigmentation, which is visible in the dorsal eye cup at approximately embryonic day 11.5 (E11.5; Strongin and Guillery, 1981). The basal side of the RPE is characterized by numerous infoldings that increase the surface area of the RPE membrane and facilitate transport and secretion (Strauss, 2005). These general characteristics of the RPE cell provide an adequate preamble to the important role this cell type serves in retinal development, physiology, and disease.

The RPE is positioned between the highly vascular choriocapillaris and the avascular photoreceptor layer of the neural retina (see figure 52.2A). Tight junctions that are present between apical surfaces of adjacent RPE cells establish the blood-retinal barrier at this location, while the endothelium is responsible for the barrier within the neural retina. Insofar as the RPE represents a barrier in the retina, one major function of this cell type is to transport water, electrolytes, and metabolic waste from the subretinal space to the choriocapillaris and import nutrients such as glucose and retinol from the systemic circulation (Strauss, 2005). Retinol (vitamin A) is important in visual sensation because it is a major source of 11-cis-retinaldehyde, the chromophore that binds opsins in photoreceptors. Light isomerizes 11-cis- retinal to all-trans-retinaldehyde, which initiates the signal transduction cascade and photoreceptor membrane hyperpolarization. The 11-cis-retinaldehyde–opsin receptor complex is restored in a process known as the retinoid visual cycle, and the RPE plays an important role in this process (Dowling, 1960).

A number of genes identified in humans and mice have been shown to encode critical components of the retinoid visual cycle, and mutations in these genes result in poor visual sensitivity and photoreceptor degeneration (Besch et al., 2003). Recent studies in mice have suggested that modulation of the retinoid cycle may be a new therapeutic strategy to treat RPE and retinal diseases. For example, the RPE65 gene encodes a protein of 533 amino acids that is required for conversion of all-trans-retinyl esters to 11-cis- retinol in the RPE (Redmond et al., 1998). Mutations that affect RPE65 activity result in Leber congenital amaurosis in humans, which is characterized by a severe, early-onset retinal dystrophy (Gu et al., 1997; Marlhens et al., 1997; Redmond et al., 2005).

The RPE65 protein is specifically expressed in RPE cells, and knockout of RPE65 in mice results in very low levels of 11-cis-retinal and its esters, while trans-retinyl esters accumulate in the RPE (Bavik et al., 1992; Redmond et al., 1998). Electroretinograms (ERGs) are severely attenuated, despite the presence of photoreceptors and outer segments (Redmond et al., 1998; Pang et al., 2005). The most obvious pathology is the shortening of outer segments in mice lacking RPE65, confirming that proteins that are intrinsic to RPE cells can often affect neighboring photoreceptors. Photoreceptor cell loss occurs in the absence of RPE65, but this event is likely secondary to biochemical changes in the visual cycle (Woodruff et al., 2003). Oral administration of 9-cis-retinal in mice lacking RPE65 led to recovery of light sensitivity and rod function (Van Hooser et al., 2000).

Administration of 9-cis-retinoid also restored visual pigment and retinal function in mice lacking the enzyme lecithin:retinol acyltransferase (LRAT), which is critical for the esterification of all-trans-retinol (Batten et al., 2005).