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

Isolation of retinal pigment epithelium or vasculature from retinas

Recent advances in analytical techniques have greatly enhanced gene expression research, and it is now possible to obtain large amounts of data from single experiments. However, separating the retinal vascular cells or RPE cells from other more prominent neuronal and glial components remains one of the main difficulties in the specific analysis of RPE or vascular gene expression in the retina. This is particularly true when using large-scale, “unbiased” techniques such as array or SAGE analysis. Although RPE and vascular components of the retina are absolutely critical to normal retinal function, the relative percentage of these cell types in the retina is low. The RPE is a monolayer, and the retinal vasculature exists as three small vascular plexuses in the inner neural retina and the single-layer choriocapillaris. Because the RPE and vascular beds constitute only a small percentage of the retinal cells, important information about gene expression in the RPE or retinal vasculature can easily be lost among genes expressed in more abundant retinal neuronal and glial cells when analyzing gene expression using RNA isolated from whole retinas.

One such study used microarray analysis to analyze gene expression during various stages of retinal vascular development by isolating mRNA from dissected whole retinas at various stages during postnatal development (Dorrell et al., 2004). Multiple genes involved in neuronal development and the transfer of visual signals also change dramatically during these developmental stages, and these genes are generally expressed at higher abundance. Thus, it is likely that many important vascular-related or RPE-specific genes remained “hidden” within background levels. Deciphering which observed changes in gene expression correlated with important roles during various stages of vascular development rather than roles in other, nonvascular retina developmental processes was difficult. Specific patterns of gene expression changes in relation to developmental progression were eventually linked to various functions such as neuronal differentiation, visual signal transfer, or vascular development (figure 56.2A) (Dorrell, Aguilar, et al., 2004). By correlating the expression profiles with potential function, an important function for R-cadherin in endothelial cell guidance during retinal vascular development emerged from this gene expression analysis study (figure 56.2B–D) (Dorrell et al., 2002). Using the large-scale gene expression analysis, R-cadherin expression was found to be correlated with major events during retinal vascular development. R-cadherin expression is high at birth and throughout the first week, when the superficial vascular plexus is actively forming. R-cadherin expression also peaks just prior to and during formation of the deep and intermediate vascular plexuses. Subsequent immunohistochemical and functional analyses demonstrated

that R-cadherin expression is localized to sites of vascular development and is required for normal vascular guidance to the characteristic retinal plexuses (see figure 56.2B–D). Many other factors are likely to be involved in retinal vascular guidance during development and maintenance of vascular homeostasis, and future gene expression studies may help further characterize these mechanisms. However, specific isolation of retinal vascular components or RPE cells would greatly enhance the use of large-scale gene expression analysis in these particular fields.

Retinal Pigment Epithelium Isolation and Gene Expression Studies Multiple strategies are currently being used to isolate RPE or vascular components from the retina for subsequent gene expression analysis. Techniques for specifically isolating RPE cells free of other contaminating retinal cells have been described. Most of these techniques use various proteolytic enzymes to dissociate the extracellular matrix, allowing the RPE to be mechanically dissected free of the choroid and underlying neural retina (Castillo et al., 1995; Mayerson et al., 1985). Because pure RPE cells can be isolated, these techniques have been valuable for specifically studying RPE gene expression in vivo, particularly gene expression in RPE isolated from diseased human retinas. In 2002, cDNA libraries from native human RPE were used to profile RPE gene expression (Buraczynska et al., 2002). This study identified several RPE-specific genes, along with multiple genes expressed in RPE as well as in other ocular and nonocular tissues. As expected, a majority of the RPE-expressed, classifiable genes included genes involved in the metabolic pathway (21%) or gene regulation and protein expression (23%). A surprisingly large percentage of expressed genes were identified as growth factors and cell signaling molecules (13%), membrane transport molecules (5%), and molecules involved in protein trafficking (7%). This is consistent with a role for RPE in maintaining retinal homeostasis through controlled cytokine expression, transport of nutrients and ions, and maintenance of the interphotoreceptor matrix (Buraczynska et al., 2002).

RPE isolation protocols have also facilitated studies that assess changes in gene expression associated with various in vivo mouse models of disease. Using large-scale microarray analysis, RPE gene expression was compared between young (2-month-old) and older (2-year-old) mice to analyze changes in RPE gene expression associated with aging (Ida et al., 2003). A number of genes, mainly involving inflammation, stress response to oxidative stress, DNA damage, and heat shock, were found to be upregulated in isolated RPE cells from older mice. Interestingly, genes involved in stimulating neovascularization were also upregulated, supporting the hypothesis that changes in RPE function related to aging may contribute to the pathogenesis of “wet” AMD (Ida et al., 2003). Few genes were found to be downregulated in

Raw expression values

Superficial vascular plexus formation

PRL

ONL

INL

IPL

GCL

Deep vascular plexus formation

Figure 56.2 A, Gene expression profile clustering of mRNA from whole mouse retinas at different postnatal developmental stages. Based on expression profile, gene function could be classified as having probable involvement in the onset of vision (mouse retinal development nears completion and vision begins around P14), neuronal development and differentiation (nears completion around P14), or potential involvement in postnatal retinal vascular development (a process that undergoes multiple changes during these

times, and thus gene expression would vary throughout). B, The expression profile of R-cadherin was determined to have a potential relationship to retinal vascular development. When R-cadherin function was blocked, the retinal vasculature failed to form normally in the superficial plexus (C ), and guidance to the normal deep vascular plexuses was disrupted (D). Vessels migrated through the photoreceptor layer and into the subretinal space. See color plate 65. (Adapted from Dorrell et al., 2002, 2004.)

this study, although the study was not comprehensive, and important downregulation of certain genes may have been missed by the stringent constraints applied during the data analysis.

Although retina dissociation and subsequent isolation of RPE cells has facilitated many important studies on RPE gene expression, one must consider the possible effects of using proteolytic enzymes to dissociate the retina. Gene expression can change rapidly on removal of cells from their native environment, an effect that occurs as extracellular matrix components are broken down or as the cells’ own membrane proteins are cleaved by enzymatic digestion. Thus, care should be taken to dissociate the retina using as gentle a proteolytic process as possible, and isolated RPE cells should be placed into RNA preservatives such as RNALater (Ambion) or Trizol (Invitrogen) quickly to minimize changes in gene expression that occur due to isolation techniques. In addition, it is critical that RPE isolation from all control and test samples is stringently performed in exactly the same manner so as not to increase the levels of technical variability discussed earlier.

Although RPE isolation is valuable for studying gene expression changes in vivo, the limited life span of normal, primary RPE cells in culture represents a substantial obstacle for cell and molecular analysis in vitro. However, the analysis of gene expression changes due to specific, controlled variables often requires in vitro studies. Several RPE cell lines have been developed that maintain many of the behavioral and physiological characteristics of primary RPE and express similar markers, such as RPE-65, cellular retinaldehyde–binding protein (CRALBP), and pigmented epithelium–derived factor (PEDF) (Kanuga et al., 2002; Lund et al., 2001). These cell lines, such as the commonly used ARPE-19 cell line, which is currently available from ATCC (catalogue no. CRL-2302), have been used in many in vitro studies to identify important RPE-related genes. Using the ARPE-19 cell line, researchers found correlative changes in gene expression following injury of the cultured monolayer. Not surprisingly, genes involved in wound repair, including DNA synthesis, DNA repair, cellular adhesion, cytokine expression, and signal transduction, were greatly upregulated during repair of the RPE monolayer. Also highly upregulated were interesting inflammatory associated genes such as mitogen-activated kinase, CD44, monocyte chemotactic protein (MCP-1), thymosin β-10, and hepatoma-derived growth factor (Singh et al., 2001). Upregulation of these genes suggests that the RPE may play a significant role during the early inflammatory response that accompanies many ocular diseases, such as proliferative vitreoretinopathy. In another study using ARPE-19 cells, various cellular adhesion molecules, growth factors, and chemokines generally involved in inflammation and repair, including MCP-1, were also greatly upregulated on vitreous

treatment of the RPE cells, a process used to mimic the repair response observed in vivo due to proliferative vitreoretinopathy (Fan et al., 2002). Time-dependent changes in gene expression on phagocytosis of rod outer segments (Chowers et al., 2004), the response of RPE to oxidative stress (Alizadeh et al., 2001; Weigel et al., 2002), RPE cell differentiation (Alizadeh et al., 2000), and many other gene expression studies have also been performed using the ARPE-19 spontaneously transformed human RPE cell line.

As with any cell line, immortalization of the RPE cells, whether spontaneous or directed, can alter function and gene expression when compared to native RPE. Using microarray analysis, significant differences were found in the gene expression profiles of cultured primary human RPE cells from four separate donors compared to ARPE-19 cells. Hierarchical clustering demonstrated that despite being obtained from a wide range of ages (48–82 years), each of the primary RPE cell groups clustered together, and no significant overlap was observed with the ARPE-19 cell lines (figure 56.3) (Cai and Del Priore, 2006). Thus, despite the definite utility of these cell lines for determining alterations in gene expression due to specific controlled changes, actual gene expression changes to similar effects may differ in vivo, and caution should be used when generalizing results from ARPE-19 cells. It should be noted, however, that no clear differences were observed in the normal (nonstimulated) expression level of genes with functions related to angiogenesis, phagocytosis, or apoptosis (Cai and Del Priore, 2006), the major RPE functions for which ARPE-19 cells are often used. Thus, ARPE-19 cells might be useful for many important studies regarding RPE gene expression, but, as with most in vitro studies, the results ultimately need to be confirmed in vivo.

The differentiation state of RPE cells, and ultimately gene expression and cellular function, can be altered by different culture conditions, particularly when grown with different

Figure 56.3 Hierarchic clustering analysis of human adult RPE from four different samples (ages ranging from 48 to 82 years) and five ARPE-19 cultures. The gene expression profiles from the four primary human samples and the five ARPE-19 samples cluster into two distinct groups with no discernable overlap, demonstrating significant differences between primary RPE and ARPE-19 cells. Thus, caution should be exercised when making conclusions of RPE gene expression from cultured, immortalized RPE cells. (Adapted from Cai and Del Priore, 2006.)

extracellular matrix components (Turowski et al., 2004). This should also be considered when using cell lines, since different levels of gene expression may be caused simply by differences in culture conditions. Another consideration is the use of specific culture conditions to help maximize similarities to RPE in vivo. For example, culture on porcine lens capsule membranes has been shown to maintain an in vivolike differentiated state in ARPE-19 cells; apical microvilli formation is induced, and rod outer segment phagocytosis properties are maintained (Turowski et al., 2004). These factors should all be considered when using cultured RPE cells for gene expression studies.

Isolation of Retinal Vasculature and Subsequent Gene

Expression Studies Isolating retinal ECs can be very difficult because much of the retinal vasculature is intricately associated with nonvascular tissue within the neural retinal layers (see figure 56.1B). Numerous EC lines are available, and isolation of primary ECs, such as human umbilical vein ECs (HUVECs), is common. However, analysis of cultured ECs is problematic because these cells, in culture, are significantly different from such cells in vivo, where the tissue microenvironment has a significant impact on the expression and downregulation of various genes. In addition, microvascular ECs from different organs, and even from different blood vessels within these organs (arterial, venous, microvascular capillaries, etc.), behave differently in response to certain stimuli such as cytokines or growth factors (Gumkowski et al., 1987). Thus, for obtaining useful retinal vascular gene expression data, the specific use of retinal microvascular ECs may be important. Several protocols for isolating retinal ECs have been published (Antonetti and Wolpert, 2003), although the isolation of retinal microvascular ECs from mice is quite difficult. In general, isolation protocols utilize proteolytic digestion of the retina, usually with solutions containing collagenase mixed with various other combinations of extracellular matrix digestion enzymes or trypsin, followed by purification of the ECs using immunoprecipitation with vascular-specific markers such as platelet-derived endothelial cellular adhesion molecule (PECAM; CD31). Few studies have been published that specifically address gene expression from retinal ECs in mouse models of disease, including no known large-scale gene expression studies to date, probably owing to the difficulty of obtaining pure primary ECs from mouse retinas. Certain retinal microvascular EC lines have been created, such as rat JG2/1 cells. These maintain many of the characteristics of primary ECs, such as expression of GLUT-1, the transferrin receptor, von Willebrand factor, and the RECA-1 antigen, high-affinity uptake of acetylated LDL, and isolectin Griffonia simplicifolia binding (Greenwood et al., 1996). However, a similar retinal EC line from mice has yet to be developed.

Many nonendothelial cells, such as astrocytes, pericytes, microglia, and perivascular macrophages, also play critical roles in both developmental and pathological neovascularization (Provis et al., 1997). Thus, studies analyzing gene expression related to changes in the retinal vasculature should also consider the role of these cells, since changes in their gene expression profiles may significantly affect retinal neovascularization. One of the main problems with isolating mouse vasculature from retinas is that the strong enzymes required to completely remove retinal ECs from the neural retina often result in EC death. Newer isolation techniques take advantage of gentle retinal dissociation techniques with lower concentrations of dissociation enzymes in an attempt to isolate small fragments of intact vessels ( J. Greenwood, pers. comm.). Although complete purification of ECs or even vessel fragments may not be achieved, the substantial enhancement of vascular-associated cells and thus vascularrelated RNA would represent an important step for retinal vascular gene expression studies. In addition, the coisolation of closely associated astrocytes, pericytes, and microglia, which remain attached to ECs during the gentle dissociation and purification methods, may actually be advantageous. This allows culture and subsequent analysis of vascular fragments rather than ECs alone, and thus is likely to more closely recapitulate the in vivo retinal microvascular environment by including multiple vascular-related cells.

Another method of isolating retinal vasculature for subsequent gene expression analysis uses laser capture microdissection technology. Using microdissection, desired cells or tissue can be isolated from frozen sections. Generally, retinal sections are fixed and the vessels are stained using an endothelial marker such as PECAM or von Willebrand factor. Prior to laser capture, the slides are dehydrated in graded ethanol solutions and dried using acetone and xylene. For most laser capture microdissection techniques, the slide must be completely dry for efficient laser-induced capture of the desired cells. Moisture results in inefficient capture of the cells and lower resolution capture, causing nearby, undesired cells to be captured as well. Because gene expression analysis requires intact mRNA, extreme caution should be used throughout dissection, sectioning of the tissue, and laser capture, and all solutions and materials should be RNase free to ensure that the RNA is not degraded. If the RNA has been protected appropriately, RNA can be subsequently extrapolated from the isolated cells using any of a number of commercial micro-RNA isolation kits (e.g., Ambion, Qiagen). Advances in technique have made RNA amplification and subsequent gene array analysis a feasible method of evaluating gene expression from limited tissue (Luzzi et al., 2003), although certain variables are inevitably added when RNA is amplified, such as incomplete amplification of mRNA ends. Laser capture microdissection and subsequent microarray gene expression analysis has already been applied

to brain microvascular studies (Liu et al., 2006), and similar studies are likely to be valuable in the retina.

Use of transgenics in gene expression analysis

The use of transgenic mice and genetic knockouts has also substantially enhanced our knowledge of gene function in the retina. As genes are identified whose expression patterns indicate a role in retinal function, the results of altering gene expression can be very informative. Analysis of mutants with altered gene expression levels, such as those that overexpress or underexpress a specific gene, or that have altered expression localization, can give important insights into a gene’s function. For example, a particular role for VEGF isoforms during retinal vascular development and pathological neovascularization was demonstrated using mice engineered to express specific VEGF isoforms. These studies demonstrated that a combination of diffusible and relatively immobile isoforms of VEGF was required for normal vascular development. Either the combined expression of VEGF120 (readily diffusible) and VEGF188 (largely immobile) (Ishida et al., 2003) or the single-factor expression of VEGF165 (Stalmans et al., 2002), which is partially diffusible through the retina, was required for normal retinal vascular development. Mice that solely expressed either VEGF120 or VEGF188 developed severely abnormal retinal vasculature characterized by abnormal vessel organization and size and inappropriate arterial and venous differentiation (Stalmans et al., 2002). Mice engineered to overexpress VEGF in the photoreceptors (by placing the VEGF gene behind a rhodopsin promoter) also developed abnormal retinal vascularization characterized by the formation of vascular sprouts that grew from the inner retinal vasculature toward the outer photoreceptor segments (Tobe et al., 1998). These studies demonstrate the utility of altered gene expression in determining functional roles for genes in retinal neovascularization.

Many genes expressed in the eye, particularly developmental genes, also have fundamental roles elsewhere that can lead to early lethality in mutants. Unfortunately, in many cases this has rendered conventional gene knockouts uninformative for analysis of specific gene functions in later stages of eye development. In addition, it is often difficult to analyze specific vascularor RPE-related roles of genes in mice with global gene deficiencies. For these reasons, conditional knockouts have become necessary to elucidate the specific role of retinal vascularor RPE-related genes. The advent of Cre/Lox technology has provided an important tool for manipulating gene expression in mice by allowing cell-specific activation or inactivation of genes. Conditional knockouts have now been created for vascular-associated cells, including ECs (Bjarnegard et al., 2004), astrocytes (Hirrlinger et al., 2006), myeloid cells (Cramer et al., 2003), and smooth muscle actin-expressing cells, such as arterial

pericytes (Regan et al., 2000). Studies using these mice have already been instrumental in advancing our understanding of the critical roles for different genes and vascularassociated cell types in general neovascularization. As cellspecific Cre mice are combined with more Lox gene knockouts, and as these mice are further exploited for retinal research, we will continue to learn more about the importance of specific gene expression in relation to retinal vascular development and disease. Cre recombinase has also recently been linked to the tyrosinase related protein-1 (Trp-1) promoter, making future RPE specific knockouts possible (Marneros et al., 2005). Although the absolute specificity of this gene promoter for RPE cells remains somewhat controversial, it should suffice for eye-specific studies, and these and future RPE specific knockouts should quickly advance the studies of RPE specific gene expression and function as well.

Correlation of retinal pigment epithelium gene expression and retinal vascularization

A strong relationship between the RPE and retinal vasculature, both during retinal development and throughout adulthood, is becoming evident. The RPE clearly induces development of the choroidal vessels. VEGF120 and VEGF165 are both expressed by mouse RPE cells, and when VEGF expression was specifically eliminated in the RPE, the choroid became absent throughout development and adulthood; scleral tissue was observed just posterior to the RPE where the choroidal vascular plexus would normally be found (Marneros et al., 2005). Although multiple retinal abnormalities were observed in these mice, including microphthalmia and an absence of Bruch’s membrane, the RPE seemed to differentiate normally, indicating that the vascular abnormalities were due to a lack of VEGFmediated pro-angiogenic signaling by the RPE. Other growth factors, such as basic fibroblast growth factor (FGF- 2), are also expressed by the RPE and have been suggested to play a critical role during normal retinal vascular development (Rousseau et al., 2000).

RPE abnormalities have also been correlated with the onset of age-related macular degeneration (AMD) (Campochiaro et al., 1999). VEGF overexpression in the RPE has been correlated with AMD progression in excised human diseased retinal tissue (Kliffen et al., 1997) as well as mouse models (Schwesinger et al., 2001; Spilsbury et al., 2000). In addition to the expression of pro-angiogenic factors that may promote choroidal neovascularization when expressed at abnormal levels, the lack of normal RPE expression of certain antiangiogenic factors may also contribute to abnormal retinal neovascularization. The RPE is known to express factors such as pigmented epithelium–derived factor (PEDF), which may help to maintain the normally quiescent retinal vasculature. PEDF expression is upregulated as retinal

vascular development is completed (Behling et al., 2002), and retinas from patients with AMD have demonstrated a lack of normal PEDF expression in the RPE compared with age-matched donors without AMD (Bhutto et al., 2006).

In addition to the definite role of the RPE during normal and pathological choroidal neovascularization, new evidence suggests that the RPE may also play an underappreciated role during development of the vascular plexuses in the inner retina. In the postnatal Balb/c mouse, vessels branch from the superficial plexus around P8 and form the deep plexus at the outer edge of the inner nuclear layer (INL). Although the precise factors that initiate this process are not known, hypoxia in the deeper retina, caused by retinal thickening, photoreceptor maturation, and the concomitant increase in neuronal activity, is likely to be a main driving force (Zhang et al., 2003). As a result, the production of growth factors in the neural retina is increased, subsequently initiating the formation of the deep vascular plexuses. As the vessels migrate toward the deep retina, the ECs must be guided to the appropriate plexuses at the outer and inner edges of the INL. Filopodial processes, which can contact and respond to preexisting guidance cues, have been observed at the tips of ECs migrating toward the deep vascular plexus (Dorrell, et al., 2002). R-cadherin expression at the outer and inner edges of the INL correlates with vascularization of the deep and intermediate plexuses, and when R-cadherin mediated adhesion is disrupted, retinal vessels migrate directly past the deep plexus, into the normally avascular photoreceptor layer and subretinal space (Dorrell, et al., 2002; Dorrell, Otani, et al., 2004) (see figure 56.2D). A similar phenotype whereby vessels invade the ONL was also observed in transgenic mice with photoreceptor-induced overexpression of VEGF (Tobe et al., 1998). This indicates that normal guidance cues can be overcome either by overexpression of growth factors beyond the normal deep vascular plexus or by masking specific adhesion molecules critical for EC guidance. Abnormal vascular sprouts invading the outer retina and the subretinal space are also observed in mice lacking the very-low-density lipoprotein receptor (VLDLR) (Heckenlively et al., 2003). Unlike the subretinal vessels that form when R-cadherin mediated adhesion is blocked, which directly bypass the deep layers, these subretinal vessels develop a few days later as sprouts from the normal deep and intermediate retinal plexuses. VLDLR is known to mediate neuronal guidance during development of the neocortex in a mechanism involving R-cadherin (Rice et al., 2001; Trommsdorff et al., 1999) and these results suggest that VLDLR expression in the retina could potentially be functioning in a similar fashion during retinal vascular guidance.

In each of these situations, retinal vessels migrate through the photoreceptor layer and into the subretinal space alongside the RPE. Although many of the growth factors that

initiate formation of the deep vascular plexuses definitely come from retinal glial and neuronal cells, these observations suggest that deep vascular sprouting may be promoted by the RPE as well. The pro-angiogenic cytokine gradient that induces development of the choroid may also participate in the development of inner retinal vascular plexuses, particularly the formation of the deep vascular plexuses. According to this hypothesis, ECs may be attracted toward the cytokine gradient formed by growth factor expression in the RPE, but normally they are guided to the deep and intermediate plexuses as they migrate. However, when these guidance cues are lost, such as the effects observed when normal R-cadherin or VLDLR function is lost, the vessels migrate past the normal deep and intermediate plexuses, through the photoreceptors, and into the subretinal space by default. More experimental evidence is certainly required to confirm or reject this hypothesis. The continued study of gene expression in both the RPE and the retinal vasculature should help answer this particular question and will continue to advance our understanding of the normal role of the RPE during normal and pathological retinal neovascularization.

Conclusion and future directions

Large-scale genomic analyses are very useful for identifying genes whose expression may be critical to various biological processes. However, gene expression analysis of the RPE and retinal vasculature is only a first step toward understanding the role these genes play in regulating normal and pathological retinal processes. As discussed in this chapter, the results of large-scale gene expression analyses need to be confirmed using other techniques, and protein expression levels must be tested before firm conclusions about the relevance of gene expression to biological function can be made. In addition, the biological effects of a particular gene’s expression, and the effects of differing expression levels in varying circumstances, must be considered. For example, many housekeeping genes, which are generally expressed at high levels, can demonstrate differential gene expression levels. However, even statistically significant differences in the expression of these genes usually have low biological relevance. Because of high expression levels and constitutive functions, even relatively substantial changes in expression may not dramatically affect retinal vascular or RPE functions. By contrast, the function of many genes critical to normal RPE and retinal vascular function, such as cytokines and growth factors, can be significantly altered by marginal differences in expression levels. Small changes in the expression of these genes may have dramatic effects leading to the onset or progression of various diseases. Thus, the biological effects of differential gene expression need to be addressed.

Finally, many genes are regulated beyond the transcriptional level. For example, HIF1-α is a critical cellular oxygen sensor. It mediates a cell’s response to hypoxia by turning on various stress response pathways and by initiating the expression of VEGF and other pro-angiogenic factors to initiate vessel growth and ultimately alleviate hypoxia. However, HIF1-α is constitutively expressed and is regulated by posttranslational degradation. Under normal oxygen conditions, HIF1-α is rapidly degraded, but in hypoxic conditions it becomes stabilized. Gene expression analysis alone would not detect the important changes in HIF1-α protein levels in cells exposed to different oxygen levels. Thus, whereas gene expression analysis is very useful, it is the amount of functional protein produced by cells that ultimately makes a difference. As techniques for detecting protein expression, such as large-scale proteomic analysis, continue to improve they will become more and more useful for the analysis of RPE and retinal vascular functions.

Since the early 1990s, gene expression analysis has seen a massive boom, largely helped by the advent of large-scale analysis techniques. As microarray technologies evolve to provide more sensitive temporal expression information, and as other techniques continue to provide information about spatial expression at the cellular level (i.e., microarray of laser-captured specimens), our understanding of normal and abnormal biological processes in the retina should be greatly enhanced. These large-scale techniques, such as microarray or SAGE, along with other more conventional techniques, such as RT-PCR and in situ hybridization, have already been instrumental in studying retinal gene expression. With the use of isolation techniques such as microdissection, specific RPE and retinal vascular gene expression analyses will continue to be possible. These studies will advance our understanding of the roles that RPE and the retinal vasculature play in normal retinal homeostasis, as well as help determine what effects alterations in normal gene expression levels might have on the progression of various ocular diseases.

acknowledgments Work was supported by the National Eye Institute (grant no. EY11254), the MacTel Foundation, Scripps Fonseca/Mericos Fund, the Robert Mealey Program for the Study of Macular Degenerations, the V. Kann Rasmussen Foundation, and the Horner Family Fund. MID was supported by a California Institute for Regenerative Medicine fellowship. We thank the entire Friedlander laboratory staff for their helpful contributions to the preparation of this chapter.

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Optimal vision depends on the transparence and refractive abilities of both cornea and lens, through which light traverses before reaching the retina. Embryonic induction, development, and postnatal maturation of the mouse lens and cornea involve a series of well-coordinated interactions between the neuroectoderm that forms the retina and the surface ectoderm that forms the lens and cornea (Chow and Lang, 2001; Cvekl and Tamm, 2004). One of the currently emerging unifying themes in developmental ophthalmology is that the transcription factors required for embryonic development of cornea and lens are also used for expression of crystallins, the specialized proteins characteristic of the differentiated states of lens and cornea, in the adult tissues. This chapter summarizes current knowledge and recent developments related to the regulation of gene expression in the mouse lens and cornea during embryonic and adult stages, with appropriate references to the human congenital eye defects associated with mutations in genes regulating eye development.

Development of lens and cornea

The lens arises by invagination of the thickened lens placode on the surface ectoderm in response to signals from the underlying optic vesicle (future retina) around embryonic day 11 (E11) in mice (figure 57.1). The anterior cells of the lens vesicle divide and remain cuboidal, while the posterior cells elongate, lose organelles, and differentiate into nondividing primary fiber cells, ultimately losing their nuclei. As development proceeds, proliferation of the anterior epithelial cells becomes progressively confined to the lens equator, where they continue to divide slowly throughout life and give rise to the cortical fiber cells. The lens cells accumulate high proportions of a few water-soluble proteins called crystallins. The transparent primary and secondary cortical fiber cells form the bulk of the lens and are responsible for its optical properties. Three major groups of crystallin gene families, α, β, and γ, each including several members, account for 90% of the water-soluble proteins in the mouse lens (Bloemendal and de Jong, 1991). It is of interest that different species often accumulate different proteins as lens crystallins,

which are known as taxon-specific crystallins (Piatigorsky and Wistow, 1991; Wistow and Piatigorsky, 1988).

The cornea has a more complex cellular composition than the lens. The surface ectoderm anterior to the lens vesicle gives rise to the corneal epithelium. Underneath the epithelium, the cornea develops a thick extracellular collagen-filled stroma littered with keratocytes and a posterior monolayer of endothelial cells derived from the neural crest (Hay, 1979; Zieske, 2004). A significant number of corneal basal epithelial cells derived from the embryonic surface ectoderm remain undifferentiated in neonatal mice and serve as corneal epithelial progenitor cells. Following eye opening, around postnatal (PN) day 15, the progenitor cells divide and stratify to form the mature cornea by about 6 weeks of age (Hay, 1979; Zieske, 2004). This proliferation and differentiation continue in the adult mouse, allowing steady replacement of the sloughed off superficial epithelial cells by the slow division of basal cells; basal cells are replenished by stem cells originating from the limbal epithelium at the periphery of the cornea (Collinson et al., 2002; Cotsarelis et al., 1989; Nagasaki and Zhao, 2003). The corneal epithelial cells and keratocytes, like the lens fiber cells, accumulate a high proportion of a few intracellular proteins, called corneal crystallins, and these often differ in different species (Jester et al., 1999, 2005; Piatigorsky, 1998). In the mouse, the corneal crystallins aldehyde dehydrogenase 3A1 (Aldh3a1) and transketolase (Tkt) account for 50% and 10% of watersoluble proteins, respectively. In addition, the corneal epithelial cells accumulate high concentrations of cytokeratins (Krt12 in the case of mice) (Tanifuji-Terai et al., 2006).

The abundance of crystallins indicates that they serve specific optical roles (known for lens, hypothesized for cornea), yet they are also expressed in lower amounts in many tissues where they have strictly enzymatic or other cellular functions. In other words, the widely expressed lens and corneal crystallins gain optical functions by virtue of their high tissue-specific expression, a situation that has been called gene sharing (Piatigorsky and Wistow, 1989; Piatigorsky et al., 1988). The gene sharing relationship between tissue-specific expression and function of a gene and its encoded protein adds an intriguing general aspect to gene expression in lens and cornea (Piatigorsky, 2007).