Molecular Vision 2007; 13:1259-73 <http://www.molvis.org/molvis/v13/a137/>Received 19 March 2007 | Accepted 20 June 2007 | Published 23 July 2007
The retinal pigment epithelium plays a central role in thephysiology of the retina [1]. As the outer-blood retinal barrier,it regulates transport between the neural retina and fenestratedcapillaries of the choroid. Among its functions, retinal pig-ment epithelium (RPE) regulates the ionic environment of thesubretinal space, phagocytizes shed outer segments of photo-receptors and participates in the visual cycle by convertingtrans-retinal to the photosensitive cis isoform. Diseases of theRPE result in retinal degeneration and degeneration of thechoriocapillaris [2-4]. Transplantation of RPE into diseasedeyes has met with limited success, perhaps because the dis-eased environment was unable to provide the environmentalsignals that regulate normal RPE function. To explore thatpossibility, our lab has studied how tissue-tissue interactionsare established during normal development to regulate RPE’sfunction as the outer blood-retinal barrier. The barrier con-sists of two components: Transcellular mechanisms regulatetransport through the cells of the monolayer and establishtransepithelial gradients. By contrast, tight junctions regulate
diffusion through the paracellular spaces, which preventstransepithelial gradients from dissipating.
Aside from an increase in melanin and minor changes inmorphology, the differentiation of RPE was thought to be com-pleted early in retinal development. More recent studies dem-onstrate changes in cell polarity, cellular metabolism and theexpression of intercellular junctional proteins [5-13]. Thesetight and adherens junctions bind the monolayer together andregulate transepithelial diffusion through the paracellularspaces. Development of the RPE can be divided into threestages that relate to the development of the inner and outersegments of photoreceptors. Across vertebrate species, thesedevelopmental milestones of the neural retina appear to belinked to developmental milestones of the choroid, Bruch’smembrane and RPE [14,15]. Previous studies related the for-mation of tight junctions to these milestones [16]. In the earlyphase, before inner segments penetrate the outer limiting mem-brane, a rudimentary adherens junction binds the RPE mono-layer together, but tight junctions are absent. Near the end ofthis phase, isolated tight junctional strands begin to appear. Inthe intermediate phase, which ends when outer segments be-gin to form, these strands gradually coalesce into a discon-tinuous network that encircles the cell. By mid-intermediatephase, the network becomes continuous and functional, but it
Analysis of the RPE transcriptome reveals dynamic changesduring the development of the outer blood-retinal barrier
Lawrence J. Rizzolo,1,2 Xiang Chen,3 Matthew Weitzman,1,2 Ru Sun,1,2 Heping Zhang3
1Department of Surgery, Yale University School of Medicine, New Haven, Connecticut; 2Department of Ophthalmology and VisualSciences; 3Department of Epidemiology and Public Health
Purpose: The morphology of the RPE shows minimal change as the neural retina and choriocapillaris differentiate.Nonetheless, initial studies of proteins related to the outer blood-retinal barrier suggest extensive remodeling of the retinalpigment epithelium (RPE) in response to this changing environment. A genomic approach was used to investigate theextent of this remodeling.Methods: RPE was isolated from E7, E10, E14, and E18 chick embryos and total RNA extracted for probing the entiregenome on Affymetrix microarray chips. Statistical parameters using ANOVA were adjusted to yield a theoretical falsediscovery rate of 5%. STEM software was used to cluster genes into statistically related patterns of expression. Geneontology clustering, using Affymetrix software was used for functional clustering. The proteinlounge.com database wasused as a source of known biological pathways.Results: Of the 37,694 probesets on the microarray, 17,199 were absent. Of the 20,495 expressed probes, the expressionof 8,889 was developmentally regulated. 4,814 of these could be clustered into 12 patterns of expression that were statis-tically significant. Minimal contamination by surrounding tissues was detected. The developmental patterns of 22 tightand adherens junction proteins were compared using hybridization to the microarray and quantitative PCR. Only twoshowed small variations from the patterns revealed by the microarray. The data indicate extensive remodeling of theextracellular matrix, cell surface receptors, cell-cell junctions, transcellular ion transport, and signal transduction path-ways throughout development. Notably, the appearance of the mRNAs for claudin 20, ZO-3, and cadherins 13 and 20 verylate in development suggest barrier properties continue to change after functional junctions are formed.Conclusions: The data reveal a far more dynamic view of the RPE and its interactions with its environment than would beexpected from morphological examination. The remodeling of junctional complexes, extracellular matrix interactions andtranscellular transport capabilities indicates a continuous remodeling of the blood-retinal barrier as the retina develops.These data provide a standard whereby culture models of RPE function and regulation may be judged.
Correspondence to: Lawrence J. Rizzolo, Department of Surgery,Yale University School of Medicine, PO Box 208062, New Haven,CT 06520-8062; Phone: (203) 785-6277; FAX: 203 785-5155; email:[email protected]
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continues to increase in depth and strand number throughoutthis period. During the late phase, the complexity (density ofanastomotic connections between strands) increases. Through-out development, progressive morphological changes were alsoobserved in the adherens junctions [17]. Throughout develop-ment, changes have been demonstrated in the expression andsteady-state levels of some proteins that form these junctionalcomplexes [6,7,12,16,18,19]. In vitro experiments indicate thechoroid and neural retina regulate this remodeling process[16,20].
The only genomic analysis of RPE development that hasbeen reported used zebrafish. That study focused on a singletime point that corresponds to the intermediate to late phasetransition [21]. To determine how extensive molecular remod-eling of the RPE might be, we used a microarray of the chickgenome to monitor how gene expression changed from theearly through the late phase of development. Approximately40% of the transcriptome that was expressed on embryonicday 7 (E7) changed by E18. The analysis focused on intracel-lular junctions and genes related to the barrier functions of theRPE.
METHODSIsolation of retinal pigment epithelium and total mRNA: Sheetsof RPE were isolated from chicken embryos on E7, E10, E14,and E18 and stored in RNAlater (Qiagen, Valencia, CA), asdescribed [16]. To isolate total RNA, the RNeasy Protect kit(Qiagen) was used according to the manufacturer’s protocols.For each age, 3-4 independent preparations were used foranalysis on Affymetrix microarrays of the chicken genome(Santa Clara, CA). For each preparation, sheets of RPE werepooled from 20-30 eyes. The quality of the total RNA wasassessed by the Keck Center, Yale University using formamidegels and a 2100 Bioanalyzer (Agilent Technologies, SantaClara, CA).
Hybridization and quantification by the reverse-tran-scriptase polymerase-chain-reaction: Hybridization of themicroarrays and initial statistical analysis for quality controlwas performed by the Keck Center at the Yale UniversitySchool of Medicine. For quantitative and semi-quantitativeassays of specific mRNAs, we used real time PCR, as de-scribed [16] with the exception that GAPDH was used in placeof 18S RNA to normalize the samples. For each age, 5-6 inde-pendent preparations of RNA were analyzed, and the standarderror is reported. The following primers were used: Claudin20, 5'-TAA CGC AGA TGC AAG GAC TG-3', 5'-GCA GACTCC TCC AGC AAA AC-3'; ZO-1 α+; 5'-AAC CCA GCAACC TCA TCA AC-3', 5'-GGA TCT ATA TGC GGC GGTAA-3'; ZO-1 α-; 5'-AAC TGC TTC TCA GCC GGT AT-3',5'-CTG CTC GTA CTC CCT ACT TGG-3'; ZO-2, 5’GAC AGGGCA GAC TTC TGG AG3',5'-TTG CCT CAC AGT GTTCAA GC-3'; ZO-3, 5'-GAC ACA AAC ATG GAC GAT GC-3', 5'-AAT GCG TCC GGA TGT AGA AG-3'. All assays wereperformed in triplicate.
Data processing: The data were first filtered to removethose probes not expressed at any time point. A probe is con-sidered to be expressed at a time point if it is detected (i.e.,
labeled as “P” by GCOS software) in at least half of the repli-cates. For those probes expressed, the raw expression signalwas log-transformed. To identify significantly differentiallyexpressed probes, we applied classic one-way ANOVA analy-sis. A probe is considered developmentally regulated if its p-value is less than or equal to a threshold that yields a theoreti-cal false discovery rate of 5% [22].
Clustering: The Short Time-series Expression Miner(STEM, version 1.2.2b) software [23] with default parameterswas used for analyzing the set of regulated probes. Briefly,STEM implements a novel clustering method that depends ona set of distinct and representative short temporal expressionprofiles and each probe in the dataset is assigned to a profilewith closest match. The expected number of probes assignedto each profile is estimated by permutation and the statisti-cally significantly over-expressed profiles are then identified.
Identification of protein pathways: Protein Lounge wasused to determine the membership of the protein pathwaysdescribed in this report. Affymetrix, Ensembl, and DAVIDsoftware were used to correlate probeset identifier numbers
Figure 1. Genes that regulate the cell cycle and are expressed byretinal pigment epithelium. The probe sets that were included in thestatistically significant clusters were sorted by the Affymetrix GeneOntology Mining Tool to identify genes that regulate the cell cycle.Many of the cell cycle genes were down-regulated as the RPE mono-layer became quiescent. Mean values of 3-4 biological replicates areindicated according to the scale, in arbitrary units, depicted at thebottom. Complete gene titles and mean values with the associated p-statistic are listed in the corresponding Appendix 2.
with the mRNA that they represent. DAVID and the Affymetrixcluster analysis tools, both based on the Gene Ontology (GO)classification system, were used to identify functional clus-ters within the statistical clusters identified by STEM soft-ware.
RESULTS & DISCUSSIONGeneral results: Of the 37,694 probesets on the
microarray, 20,495 were detected and 8,889 of those repre-sented genes that were developmentally regulated (for raw datasee the Gene Expression Omnibus, GSE7176). Using STEMsoftware, the regulated probesets were clustered into groupsaccording to their pattern of expression. Of the 50 clusters, 12were judged to be statistically significant, and potentially con-tain genes that are coordinately regulated. These 12 clustersincluded 4,814 probesets. Expression increased for 6 clustersthat could be distinguished according to the phases of devel-opment in which increases occurred (early: E7-E10, interme-diate: E10-E14, and/or late: E14-E18). Similarly, expressiondecreased in the remaining 6 clusters. In the descriptions thatfollow, genes were grouped according to whether the expres-sion of the mRNAs was stable over time, increased, decreasedor were regulated but not included in a statistically significantcluster. Memberships and a graphic representation of the sta-tistically significant clusters are included in the Appendix 1.
The validity of these findings would be compromised ifthe neural retina or the choroid contaminated the preparationsof RPE. To minimize contamination, the RPE was isolated inlarge sheets that were readily distinguished by their pigmentfrom the neural retina and choroid. Several lines of evidencesuggested contamination was minimal. We examined 6 chor-oidal markers and 12 retinal markers. Only 4 were detected in
Figure 2. Visual cycle genes expressed in retinal pigment epithelium.The Protein Lounge database was used to identify cycle members.Some mRNAs for visual cycle proteins increased throughout devel-opment, others only during the late phase when photoreceptor outersegments are forming. The apparent variation of interphotoreceptorretinoid binding protein (IRBP) was statistically insignificant. Lowsignals with large statistical errors typified the few genes that are notexpressed by retinal pigment epithelium, but were detected on themicroarray. Mean values of 3-4 biological replicates are indicatedaccording to the scale, in arbitrary units, depicted at the bottom. Com-plete gene titles and mean values with the associated p-statistic arelisted in the corresponding Appendix 2.
Figure 3. Phagocytic pathway genes expressed in retinal pigmentepithelium. The Protein Lounge database was used to identify path-way members in macrophage. Some mRNAs for phagocytic pro-teins increased throughout development, others only during the latephase when photoreceptor outer segments are forming. Mean valuesof 3-4 biological replicates are indicated according to the scale, inarbitrary units, depicted at the bottom. Complete gene titles and meanvalues with the associated p-statistic are listed in the correspondingAppendix 2.
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our preparations. These were expressed at low levels, had largestandard deviations, did not vary with age, or were detectedon ages when they are not expressed in vivo [24]. For thechoroid, the endothelial cell markers: Fli-1, CD144 (VE-cadherin), vascular cell adhesion molecule (VCAM) and CD34were undetected. Further, the neural cell adhesion molecule(NCAM), a prominent component of chick choroid, but not ofchick RPE, was undetected [13]. However, the endothelial cellmarker, CD31 (PCAM) was evident. For the retina, undetec-ted markers included opsin-1 (iodopsin), opsin-3 opsin-4, op-sin-5, rhodopsin (opsin 2), rhodopsin kinase, and several cy-clic nucleotide-gated channels. Although violet and blue coneopsins were detected, they were also evident on E7, when theywere not detected by PCR [24]. These data suggest that somelow hybridization signals may reflect non-specific binding andindicate that the RNA preparations were minimally contami-nated with the surrounding tissues.
Correspondence to anticipated and known patterns of ex-pression: Early differentiation and withdrawal from the cellcycle: Another way to validate the data is to examine patternsof expression that would be anticipated from the literature.The differentiation of the outer layer of the optic cup into RPEis initiated by the interaction of three transcription factors,Pax6, MitF and Otx2 [25-27]. Pax 6 expression decreases inthe RPE during development and becomes restricted to theneural retina. In contrast, Otx2 is specific for the RPE. All
three were detected on the microarray even though the E7 timepoint is after differentiation has begun. By contrast, Chx10, anegative regulator of RPE differentiation, and Rx1, an initia-tor of ocular development were not detected, indicating mini-mal contamination by the neural retina. The hybridization sig-nal for Pax6 decreased 3x (p<0.0001) and MitF decreased 2x(p<0.002) between E7 and E18. As would be expected, Otx2increased 1.5x (p<0.03). We were able to determine smallchanges with statistical confidence, because the ANOVA en-compassed all 4 time points. As discussed at the end of thissection, these small changes detected by hybridization trans-lated into large changes, as measured by quantitative RT-PCR.Altogether, 47 transcription factors and regulators were foundto decrease expression during development, while 48 othersincreased (Appendix 1).
Proliferation of the RPE decreases substantially in thecentral zone by E5 and by E12 is very low around the entireglobe [28]. Accordingly, one would expect a decrease in theexpression of genes that promote progression through the cellcycle and an increase in those that repress it. Figure 1 includes30 cell-cycle genes that were developmentally regulated; 21decreased. Of the nine than increased, proteins such as pro-tein phosphatase 1, cyclin D1 and Cdc42 participate in a vari-ety of cellular processes [29-31].
Visual cycle: The proteins of the visual cycle might beexpected to increase in expression about the time that outer
Figure 4. Lysosomal proteins expressed in retinal pigment epithe-lium. The Protein Lounge database was used to identify lysosomalproteins. Some mRNAs for lysosomal proteins increased throughoutdevelopment, others only during the late phase when photoreceptorouter segments are forming. Mean values of 3-4 biological replicatesare indicated according to the scale, in arbitrary units, depicted at thebottom. Complete gene titles and mean values with the associated p-statistic are listed in the corresponding Appendix 2.
TABLE 1. COMPARISON OF HYBRIDIZATION TO THE MICROARRAY WITH
REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION
Reference for PCR mRNA E7-E10 E7-E10 E7-E10 analysis----------- ------ ------ ------ -----------Claudin 1 + + + [16], This reportClaudin 2 + + + [16]Claudin 3 NDE NDE NDE [16]Claudin 4 NDE NDE NDE [16]Claudin 4L2 + + + [16]Claudin 5 + + + [16]Claudin 10 HMA HMA HMA [16]Claudin 11 + + + [16]Claudin 12 + + + [16]Claudin 15 NDE NDE NDE [16]Claudin 20 + + + This reportJAM-B NDE NDE NDE [18]JAM-C NDE NDE NDE [18]Par 3 + + - [18]Par 6 + + + [18]AF-6 - + + [18]ZO-1 + + + This reportZO-2 + + + This reportZO-3 + + + This reportGLUT 1 + + + [71]GLUT 2 NDE NDE NDE [71]GLUT 3 + + + [71]
The change in expression between the indicated ages was measuredby quantitative or semi-quantitative RT-PCR or by hybridization tothe microarray. Agreement between the two methods is indicated bya plus (+); disagreement is indicated by a minus (-). NDE indicatesnot detected by either method. HMA indicates only detected by hy-bridization to microarray.
segments are made [8,32]. For some visual cycle proteins, theincrease in mRNA was gradual, but for lecithin retinolacyltransferase and retinol dehydrogenase 12 a major increasewas observed in the latter phases of development (Figure 2).The largest increase was for RPE65 (50x). Similar large in-creases in the late phase for other enzymes might have beenobscured, because the microarray was saturated. There ap-peared to be stable expression of the mRNA for theinterphotoreceptor retinoid binding protein (IRBP), a carrierprotein for retinoids that is secreted in to the subretinal space.In contrast to the other mRNA’s, the signal for IRBP was lowand the standard error was large. IRBP might be expressed by
chick RPE, as it is in zebrafish [33]. It is more likely that lowsignals with large errors were the hallmarks of non-specificbinding or minor contamination by the neural retina.
Phagocytosis: Lysosomal proteins and phagocytic path-ways should be expressed throughout development, but in-creased expression might be expected in the late phase whenouter segments are being formed and RPE microvilli are length-ening in response. Shedding of outer segment discs would in-crease the phagocytic load on the RPE and there would be aneed for membrane receptors (and associated signaling path-ways) that are specific for the outer segments. Sixty sevengenes associated with phagocytosis were examined (Figure
Figure 5. Correspondence of hybridization to the microarray and quantitative polymerase chain reaction of claudin mRNAs. The left panelsshow PCR data taken from Rahner et al. [16], reproduced with permission. The data for claudin 1 was re-evaluated in this study to address thelarge error for E18 in the original study. The right panels show the corresponding data from the microarray. The standard error is shown whenit exceeded the size of the symbol. Qualitatively, both methods revealed the same pattern and relative level of expression. Quantitatively,hybridization was less sensitive to small changes. The small, but statistically significant, increase in claudin 4L2 that was measured byhybridization was observed to be a 35x increase when measured by PCR.
3). The expression of 60% of these was developmentally regu-lated and most of these increased their expression of mRNA.Notably, the mRNAs of receptor proteins for shed outer seg-ments, CD36 and integrin subunit αv, increased during devel-opment. Although the magnitude of the changes was not asgreat, a similar pattern was observed for the 25 lysosomal en-zymes that were examined (Figure 4).
Correlation of hybridization to the microarray with quan-titative PCR: Table 1 summarizes the comparison of the twomethods. With the exception of claudin 10, both methodsagreed on whether an mRNA was present or absent. The nextcomparison examined whether the methods agreed that sig-nificant changes did, or did not, occur between the E7 to E10,E10 to E14 and E14 to E18 time points. Hybridization failedto detect a 7x decrease in AF-6 mRNA between E7 and E10 ora 5x increase in Par 3 mRNA between E14 and E18 [18]. This
is consistent with the observation that a 35x increase in claudin4L2, measured by PCR, registered as only a 1.7x increase onthe microarray (see Figure 5). JAMs B and C were not de-tected by either method (JAM-A was not represented in themicroarray). The biological significance of these data is dis-cussed in the next section. These limited data demonstrate ahigh correlation between the qualitative patterns of expres-sion exhibited by the hybridization and PCR techniques (Table1). The presence or absence of only 1 of these 22 mRNAs wasmisidentified by hybridization. When neighboring time-pointswere compared, relative expression was misrepresented onlytwice out of 45 comparisons. It is unclear why hybridizationto the microarray was insensitive to changes <7x, as measuredby PCR. This insensitivity illustrates the power required ofthe statistical methods, as small changes measured on themicroarray reflected changes large enough to be of biological
Figure 6. Correspondence of hybridization to the microarray and quantitative polymerase chain reaction of ZO and claudin 20 mRNAs. Thehybridization data are represented by black circles. For ZO-1, the hybridization data measure total ZO-1 mRNA, whereas the polymerasechain reaction (PCR) data are specific for ZO-1α+ (red squares) or ZO-1α- (green diamonds). For ZO-2, ZO-3, and claudin 20, the PCR dataare represented by red squares. Qualitatively, both methods revealed the same pattern and relative level of expression. The data for ZO-1indicates that the splicing of its hnRNA was developmentally regulated. Hybridization to microarray, left y-axis; Quantitative PCR, right y-axis.
significance. By using ANOVA to analyze the entire time-course, it was possible to make the small distinctions includedin the following figures with a theoretical false discovery ofrate 5% [22]. P-values for the figures are included in the Ap-pendix 2.
These data indicated the mRNA preparations and statisti-cal methods were of sufficient quality to examine the mRNAsfor the proteins that enable RPE to interact with its environ-ment, regulate its morphology and function as a blood-retinalbarrier. The time-course of expression focused attention ongenes that were both developmentally regulated and membersof clusters whose pattern of expression was statistically cor-related [23]. The next section further verifies and quantifiespredictions based on hybridization to the microarray by using
quantitative, real-time PCR of tight junctional proteins.Remodeling of the apical junctional complex: Barrier
function requires an apical junctional complex composed ofadherens and tight junctions. The complex allowstransepithelial concentration gradients to form and transmitssignals that regulate cell size, shape, polarity and prolifera-tion [34-39]. The transmembrane proteins of the complex arelinked by adaptor proteins to filamentous actin and a varietyof effector proteins [38]. The gradual remodeling of the com-plex during RPE development has been described at the mor-phological and molecular level for the adherens [6,7,17] andtight [12,16,18,19] junctions. The current study revealed al-terations in gene expression that imply more extensive modu-lation of the complex’s signaling capacity than was previouslyrecognized.
Tight junctions: Earlier studies on the development of RPEtight junctions focused on the claudins. The claudin family oftransmembrane proteins form the tight junctional strands ob-served by freeze-fracture electron microscopy and determinethe selectivity of the paracellular pathway [40]. Changes inthe expression of the claudins correlated with morphologicalchanges in the fine-structure of RPE tight junctions. Severalclaudins were regulated at the level of transcription, proteinstability and subcellular localization [16]. The qualitativeagreement between PCR and hybridization that was noted inTable 1 is shown in detail in Figure 5 and Figure 6. Claudins3, 4, and 15 were not detected by either method, but a weaksignal was detected for claudin 10 on the microarray. Theclaudins that were detected by both methods exhibited verysimilar developmental patterns. The microarray revealed anadditional claudin not previously reported for RPE, claudin20. A large increase in claudin 20 mRNA occurred late in de-velopment, which was confirmed by real-time RT-PCR (Fig-ure 6). By E18, the copy number for claudin 20 mRNA was
Figure 7. Tight junction genes and known regulators expressed inretinal pigment epithelium. The Protein Lounge database was usedto identify members. Close to 50% of the genes identified were de-velopmentally regulated. Although tight junctions are forming dur-ing this period, expression of a number of genes was down-regu-lated. Several genes, such as ZO-3 and claudin 20, increased prima-rily after E14 when there are few structural changes in the junction.Mean values of 3-4 biological replicates are indicated according tothe scale, in arbitrary units, depicted at the bottom. Complete genetitles and mean values with the associated p-statistic are listed in thecorresponding Appendix 2.
Figure 8. Adherens junction genes and associated regulatory pro-teins expressed in retinal pigment epithelium. The Protein Loungedatabase was used to identify members of this class. Close to 50% ofthe genes identified were developmentally regulated. The data sug-gest extensive remodeling, as the expression of many genes was down-regulated even as expression of others was up-regulated. Genes forseveral cadherins were detected that were not previously associatedwith RPE junctions, including H-cadherin and cadherin 20. Meanvalues of 3-4 biological replicates are indicated according to the scale,in arbitrary units, depicted at the bottom. Complete gene titles andmean values with the associated p-statistic are listed in the corre-sponding Appendix 2.
greater than claudin 1, which suggests these are the most promi-nent claudins in the RPE tight junction. The magnitude of thislate-phase increase was surprising given the minimal changesin the fine-structure of the tight junctions [16,41]. By con-trast, the expression of claudins 1 and 5 did not increase afterE14 (Figure 5). Although the mRNA for claudins 2 and 4L2did increase in the late phase, their level of expression waslower than claudins 1 and 20. Nonetheless, minor claudinscan modulate the function of the RPE tight junction [42-44].Although the steady-state level of claudin 20 protein and itssubcellular distribution remain to be determined, the chang-ing ratio of the claudin mRNAs during development impliessignificant changes in function. This maturation of functionoccurred after the structure of the tight junctions was estab-lished [16], but during the time photoreceptors matured withthe formation of outer segments.
The transmembrane proteins of the tight junction arelinked to effector proteins and the cytoskeleton by adaptorproteins [38]. We focused on the ZO family of adaptors. ChickRPE expresses ZO-1 and ZO-1 like protein (ZO-1LP), whichby M
r, immunogenicity and ability to bind occludin appear to
be the orthologs of mammalian ZO-1α- and ZO-1α+ [12,19].The difference between the two variants is an internal sequenceof 80 amino acids, the alpha region. ZO-1α+ is more specificfor epithelia. In RPE, ZO-1α- was expressed at high steady-state levels in the early phase and steadily declined duringdevelopment. By contrast, ZO-1α+ protein was absent duringthe early phase but increased until E12 to become the domi-nant ZO-1 variant, whereupon its expression also decreased[19]. Unlike the proteins, the microarray data indicated thatthe expression of ZO-1 mRNA was constant (Figure 6). Toconfirm its presence in chick, primers that span the alpha re-gion were used to subclone and sequence it. RT-PCR con-firmed that ZO-1α- and ZO-1α+ mRNAs were expressed inRPE throughout development (data not shown). To pursue thisfinding, primer pairs were synthesized that were specific foreither ZO-1α- or ZO-1α+. Although the ZO-1α- variant wasconstant, the ZO-1α+ variant increased throughout develop-
ment (Figure 6). These data suggest two mechanisms that regu-late expression of ZO-1. Regulation of RNA splicing wouldlead to a change in the ratio of ZO-1α- and ZO-1α+, and regu-lation of translation or degradation would affect each splicevariant equally to decrease the steady-state protein levels ob-served during development.
Like ZO-1, steady-state levels of ZO-2 fell during devel-opment [12], even though ZO-2 mRNA did not decrease (Fig-ure 6). By contrast, both hybridization and PCR detected anincrease in ZO-3 mRNA during the late phase, between E14and E18. This paralleled the late-phase increases of claudins2, 4L2 and 20 that occur after the morphology of the tightjunction is established [16].
In the early stage, there are few tight junctional strandsdespite the presence of many proteins implicated in the as-sembly of tight junctions, such as JAM-A, AF-6, PAR 3 and6, occludin (not represented on the array), ZO-1 and ZO-2[12,18,19]. This list of pre-existing proteins appears to be moreextensive, given the mRNAs identified in Figure 7. Notably,the mRNAs for the adaptor proteins MAGI 1 and MUPP1decreased, but for the adaptors MAGI 2 and MAGI 3, mRNAswere stably expressed.
It is counter-intuitive that assembly and adaptor proteinsof the tight junction decrease when tight junctions are form-ing. This counter-intuitive behavior can be understood in termsof the assembly of the entire apical junctional complex. Manyof these proteins also assemble the adherens junction. Theyfirst form a primordial complex that reorganizes to segregatedifferent proteins into an adherens and a tight junction [45-50]. Unlike these experimental models, RPE differentiation isspread over a long period of time, which facilitates a moredetailed examination of the remodeling process. In RPE, manyof the proteins required to make a tight junction are present inadvance, in association with an adherens junction, awaitingthe appearance of the claudins and ZO-1α+. When the apicaljunctional complex remodels, the entire complex apparentlyrequires less of these adaptor and assembly proteins. By con-trast, the mRNA for other adaptor proteins, MAGI 2 and 3,are stably expressed and ZO-3 increases after assembly of tightjunctions appears to be morphologically complete. Betweenhuman and chick, ZO-3 is least conserved of the ZO proteins
Figure 9. Genes for laminin subunits expressed in retinal pigmentepithelium. Mean values of 3-4 biological replicates are indicatedaccording to the scale, in arbitrary units, depicted at the bottom. Thesechanges in expression would enable the RPE to replace embryoniclaminins with adult laminins in the basal lamina. Complete gene titlesand mean values with the associated p-statistic are listed in the corre-sponding Appendix 2.
TABLE 2. LAMININS INFERRED FROM FIGURE 9
Potential laminin mRNA detected------------------ --------------------------type composition α1 α3 α5 γ1 β2 γ1---- ----------- -- -- -- -- -- -- 1 α1β1γ1 s d s 3 α1β2γ1 s s s 6 α3β1γ1 s d s 7 α3β2γ1 s s s 10 α5β1γ1 i d s 11 α5β2γ1 i s s
Potential laminins were inferred based on the subunits that were de-tected Figure 9. Abbreviations: s indicates stably expressed; i indi-cates increased expression with embryonic age; d indicates decreasedexpression with embryonic age.
and is not functionally redundant with ZO-1 and ZO-2[15,51,52]. Therefore, the assembly and maturation of theapical junctional complex appears to involve a condensationof diverse proteins into a primordial complex, followed by aprocess of segregation and pruning, as new proteins are addedand others are reduced to mature levels. Because these adap-tor proteins express a large number of protein binding sites[15,53,54], and because the transcription of affiliated effectorproteins are also transcriptionally regulated (Figure 7 and Fig-ure 8), it is reasonable to assume that all the diverse functionsof the apical junctional complex are modulated during devel-opment.
Adherens junctions: The remodeling of the adherens junc-tion during chick RPE development has been documented bymorphology and changes in the expression of the cadherins[6,7,17]. The expression of N-cadherin decreases throughoutdevelopment, R-cadherin peaks at E14, and B-cadherin de-
creases after E14. In the current study, these changes weremirrored by the changes in mRNA levels (Figure 8). How-ever, the expression of additional cadherins was observed. AsB-cadherin expression declined in the late phase, cadherin-13(heart cadherin) and cadherin-20 each increased 5X(p<0.00001). In other tissues, cadherins 11 and 13 are regu-lated in opposite directions, with cadherin 11 more typical ofmesodermal tissues [35,55]. In RPE, expression of cadherin11 decreased only transiently (p<0.00003). Cadherin 20 hasbeen reported in melanocytes and neuroepithelial tissues, in-cluding the optic vesicle in mice [56]. Consistent with thischange in receptors, there was regulation of members of theirsignaling pathways, such as Fyn, Zyxin and Lef1 (Figure 8).
Remodeling of the extracellular matrix: The modulationof the apical junctional complex occurs in the context of dra-matic environmental changes and alteration of the RPE’s abilityto respond to those changes [15,16,20,37]. Besides knowneffects on the retina and choroid, the RPE modulated compo-nents of its extracellular matrix. The mRNA for the lamininchains required to form laminin 3 (α1β2γ1) and laminin 7(α3β2γ1) were present throughout development (Figure 9 andTable 2). Laminin 1 (α1β1γ1) presumably decreased because
Figure 10. Genes for collagen subunits expressed in retinal pigmentepithelium. These changes in expression would enable the RPE toreplace embryonic collagen IVs with adult collagen IVs in the basallamina. The other changes in collagen expression might contributeto the maturation of the collagenous layers of Bruch’s membrane.Mean values of 3-4 biological replicates are indicated according tothe scale, in arbitrary units, depicted at the bottom. Complete genetitles and mean values with the associated p-statistic are listed in thecorresponding Appendix 2.
Figure 11. Extracellular matrix receptors and associated protein genesexpressed in retinal pigment epithelium. The probe sets that wereincluded in the statistically significant clusters were sorted by theAffymetrix Gene Ontology Mining Tool to identify relevant genes.The complement of plasma membrane receptors changed during de-velopment, which suggests a capacity to respond to the new signalsthat would be expected from the differentiating neural retinal andchoriocapillaris. Mean values of 3-4 biological replicates are indi-cated according to the scale, in arbitrary units, depicted at the bot-tom. Complete gene titles and mean values with the associated p-statistic are listed in the corresponding Appendix 2.
the mRNA for the β1-chain decreased throughout develop-ment. By contrast, laminin 11 (α5β2γ1) presumably increasedbecause the mRNA for the α5-chain increased throughoutdevelopment. During the intermediate and late phases, therewas also an increase in the expression of the β3-chain. How-ever, one of its partners, the γ2-chain, was not detected. There-fore, the significance of β3 (and βx) remains to be explained.These changes represented a shift from an embryonic matrixenriched in laminin 1 chains to a more mature matrix that con-tains laminins 3, 7, and 11.
The transition from an immature to a mature basal laminawas also evident in the expression of collagen IV mRNAs(Figure 10). Six isoforms have been reported for the α-chainof collagen IV. The α6-chain, and the embryonic α2-chain
decreased during development, but this was countered by anincrease in the α4 chain (found in differentiated basal lami-nae). The α5 chain of collagen 4 appeared to be regulated, butit was not a member of any of the statistically significant clus-ters. Together with the changes in laminin mRNA, there wasthe potential to form a basal lamina that resembled the spe-
Figure 12. Actin, myosin and associated protein genes expressed inretinal pigment epithelium. Relevant transcripts were identified byusing the Affymetrix Gene Ontology Mining Tool to sort probe setsthat were included in the statistically significant clusters. Thesechanges in expression occurred when the cytoskeleton would be re-modeled to accommodate microvilli that are elongating, phagocyticpathways that are maturing and cortical actin that is remodeling tointegrate with the forming tight junctions. Mean values of 3-4 bio-logical replicates are indicated according to the scale, in arbitraryunits, depicted at the bottom. Complete gene titles and mean valueswith the associated p-statistic are listed in the corresponding Appen-dix 2.
Figure 13. Regulators of actin expressed in retinal pigment epithe-lium. Relevant mRNAs were identified by using the Affymetrix GeneOntology Mining Tool to sort probe sets that were included in thestatistically significant clusters. These changes in expression occurredwhen the cytoskeleton would be remodeled to accommodate mi-crovilli that are elongating, phagocytic pathways that are maturingand cortical actin that is remodeling to integrate with the formingtight junctions. Mean values of 3-4 biological replicates are indi-cated according to the scale, in arbitrary units, depicted at the bot-tom. Complete gene titles and mean values with the associated p-statistic are listed in the corresponding Appendix 2.
1268
cialized basal laminae of lung, amniotic and placental mem-branes [57,58]. Notably, amnionic membranes and an extra-cellular matrix derived from placenta appeared to promote RPEdifferentiation in other species [59-62], whereas a tumor-de-rived, embryonic-like matrix failed to support normal differ-entiation [63].
The mRNAs for various other collagens were evident.These were presumably destined for the collagenous layers ofBruch’s membrane that form in the latter stages of develop-ment. Evidence was found for the stable expression of col-lagen types 7, 9, 17, 18, and 20. The expression of types 1, 5,and 11 appeared to decrease during development, whereastypes 3, 6, and 8 increased.
Remodeling of surface receptors: Receptors and signal-ing pathways determine how the RPE would respond to itsremodeled environment. Matrix receptors, and their intracel-lular binding partners, shifted their expression (Figure 11).For example, integrins are αβ dimers. The mRNAs for theα1, α2, α6, and α5 integrin chains were stably expressed, butthere was a decrease for α8 and β1. The latter mRNAs werereplaced by α7, αv, and β8. The significance of these changesis best understood for integrin αvβ5 and CD36, which signalthe phagocytosis of shed photoreceptor outer segments [64,65].The significance of the other changes noted in Figure 11 re-mains to be investigated, but likely forms the basis for thedifferential responsiveness E7 and E14 RPE to environmentalstimuli. In culture, E7 RPE responds to different retinal fac-tors than E14 RPE [66]. Retinal condition medium affects theexpression of some claudin family members in E7 RPE, butother members in E14 RPE [16].
The apical junctional complex and extracellular matrixreceptors activate signal transduction pathways. Gene ontolo-
gies were used to identify signal transduction pathways thatwere represented in the statistically significant clusters. Sev-eral identified pathways related to RPE differentiation. In theTGFβ family, activin receptor type I mRNA increased duringthe early phase 1.2x (p<0.005), although activin receptor IIBmRNA decreased 5.3x (p<0.00001) throughout development.Activin βB (5x, p<0.0009) and BMP2 (1.4x, p<0.0001)mRNAs increased during the early phase. Similarly, a num-ber of wnt and frizzled (wnt receptors) mRNAs were devel-opmentally regulated. Altogether the expression of 175 genesinvolved in signal transduction changed during this period ofdevelopment (Appendix 2).
The junctional complexes and many extracellular matrixreceptors interact with the cytoskeleton. Actin and myosin areimportant regulators of apical junctional complex [67-70].During RPE development, regulated expression was observedfor the mRNA of 40 actin-myosin associated proteins (Figure12). The distribution and function of the actin-based cytoskel-eton is regulated by small G-proteins and their effector pro-teins. The mRNA for 51 proteins of this class were identifiedby the microarray, with the expression of 60% regulated dur-ing development (Figure 13).
Microtubules help mediate the intracellular trafficking ofvesicles, the polarized distribution of proteins and phagocyto-sis. The expression of mRNA for 13 microtubule related pro-teins were regulated during development (Figure 14). Therewas very little change in the expression of intermediate fila-ments. The predominant intermediate filament expressed inchick RPE is vimentin, which was stably expressed in thisstudy. Evidence was found for keratins B4, 14, and 15, glialfibrillary acidic protein, restin and desmuslin. The only inter-mediate filament whose expression was regulated was restin,which increased 2.1x (p<0.02) in the intermediate and latephases.
Transcellular components of the blood retinal barrier:
Figure 14. Microtubule associated protein genes expressed in retinalpigment epithelium. Relevant mRNAs were identified by using theAffymetrix Gene Ontology Mining Tool to sort probe sets that wereincluded in the statistically significant clusters. These changes inexpression occurred when the cytoskeleton would be remodeled tomediate increase intracellular trafficking of phagosomes, secretoryvesicles and melanin migration. Mean values of 3-4 biological repli-cates are indicated according to the scale, in arbitrary units, depictedat the bottom. Complete gene titles and mean values with the associ-ated p-statistic are listed in the corresponding Appendix 2.
Figure 15. Glucose transporter genes expressed in retinal pigmentepithelium. Expression of the mRNAs for 4 of the facilitated glu-cose transporters (SLC2A_) increased as tight junctions were form-ing to close the paracellular pathway to glucose. A Na+-coupled glu-cose transporter (SLC5A1) also increased during this period. Meanvalues of 3-4 biological replicates are indicated according to the scale,in arbitrary units, depicted at the bottom. Complete gene titles andmean values with the associated p-statistic are listed in the corre-sponding Appendix 2.
An examination of the outer blood-retinal barrier should alsoconsider the transcellular transport pathway. The transcellularpathway also develops gradually. As tight junctions form, theparacellular pathway for transepithelial glucose transportcloses, which requires RPE to increase glucose transportthrough the transcellular pathway. Previous studies indicatedthat the housekeeping transporter, GLUT3 (CEF-GT3) wasstably expressed, but that GLUT1 (SLC2A1) increased dur-ing development [71]. These data were confirmed and extendedby the current study (Figure 15), but the story proves to bemore complicated. There was stable expression of the mRNAfor GLUT 10 (SLC2A10), but continuous or late phase in-creases for GLUT 8, 9, and 11 (SLC2A8, 9, 11). These in-creases corresponded to the time when functional tight junc-tions are forming. Besides these facilitated transporters, themRNA for a sodium-coupled glucose transporter, SGLT1(SLC5A1) appeared on E10 and increased 2.5x by E18. Thistype of transporter has not been previously reported in RPE.Together, these data indicate several mechanisms that wouldenable RPE to satisfy the retina’s need for glucose after tightjunctions become functional.
The RPE establishes a polarized distribution of ion pumps,channels and transporters to mediate active transcellular trans-port of ions and organic solutes. Transport physiologists havedetermined what types of transporters must exist in the apicaland basolateral membranes of RPE, and many of these havebeen identified [72,73]. The microarray data suggested a morecomplicated story. Figure 16 lists the types of transport pro-teins identified by physiologists and the mRNA for candidatesrepresented in the chick transcriptome. There were often mul-tiple candidates for a given transporter. Further for a giventransporter, expression of one candidate might be up-regulatedwhen another candidate might be down-regulated or stablyexpressed. The data also suggested more transporters than havebeen characterized by the transport physiologists. Further in-vestigation is needed to understand how this remodeling oftransporter expression affects function and relates to the chang-ing ion selectivity of the tight junctions.
A few observations are of note. In contrast to most epi-thelia, the Na,K-ATPase is partially polarized towards the api-cal membrane, and there is some suggestion that the beta sub-unit may be important for this atypical localization [74-76].The β1 subunit mRNA was stably expressed, but the α1 andβ2 subunit mRNAs increased during the intermediate and latephases, when microvilli were extended and the distribution ofthe ATPase became polarized [11]. Bestrophin, a member of anovel class of chloride channel, is found in the basolateralmembrane of RPE [77]. Genetic defects in bestrophin resultin Best vitelliform macular dystrophy. The mRNA forbestrophin increased during the intermediate and late phasesof development. The monocarboxylate transporters are im-portant for transporting lactate out of the retina [5,78]. A po-larized distribution has been described for family members 1and 3. The apical family member (member 1) increased in theintermedia and late phases, but the basolateral family member(member 3) decreased. The story becomes more complicated,as the microarray identified 3 additional players (family mem-
Figure 16. Ion transport protein genes expressed in retinal pigmentepithelium. Transporters are grouped according to their subcellularlocation, as reported in recent reviews [72,73]. Additional transport-ers of the plasma membrane were also detected and are included atthe bottom of the figure. For many of the transporters predicted byphysiologists, a number of candidates were identified. Surprisingly,candidates with similar functions sometimes replaced one anotherduring development. An example is the Ca++-ATPase, ATP2B1,mRNA decreased as the ATP2B2 mRNA increased. Mean values of3-4 biological replicates are indicated according to the scale, in arbi-trary units, depicted at the bottom. Complete gene titles and meanvalues with the associated p-statistic are listed in the correspondingAppendix 2.
bers 5, 8, and 12), two of which increased as the other de-creased during development. The polarized distribution ofthese 3 family members remains to be determined.
The molecular remodeling of the RPE is far more exten-sive than ever imagined. As the neural retina andchoriocapillaris differentiate, the RPE demonstrates its capacityto remodel the extracellular matrix and to update the signaltransduction pathways that respond to the new signals that theapical and basal environments will provide. As the demandsof the differentiating neural retina change, the RPE appears toreformat its functions as the outer blood-retinal barrier. ThemRNAs are expressed for a new complement of transmem-brane transporters and for proteins that would alter the speci-ficity of its tight junctions. Although not the focus of this re-port, it stands to reason that there are corresponding changesin metabolic and catabolic pathways. There are several limi-tations to this analysis. The annotation of the chick databaseis a work in progress, and the microarray data must be consid-ered provisional until confirmed by more rigorous methods.Further, studies of protein expression and subcellular distri-bution are needed for a more complete understanding. None-theless, the hypotheses generated by this analysis give a di-rection to further studies. Hopefully, this overview will en-courage investigators to mine the data set to address additionalfunctions of the RPE.
ACKNOWLEDGEMENTS The Authors thank Drs. Yan Luo and Masayuki Fukuhara forhelp with preliminary studies. This work was supported byNational Institutes of Health grant EY08694 (LJR) and COREgrant EY00785 (Department of Ophthalmology and VisualScience, Yale University).
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The appendices are available in the online version of this article at http://www.molvis.org/molvis/v13/a137/.
The print version of this article was created on 23 Jul 2007. This reflects all typographical corrections and errata to the article through that date.Details of any changes may be found in the online version of the article. α
