Molecular Vision 2004; 10:588-597 <http://www.molvis.org/molvis/v10/a71/> Received 22 January 2004 | Accepted 10 August 2004 | Published 24 August 2004

Download Reprint

Early transcriptional changes of retinal and choroidal TGFβ-2, RALDH-2, and ZENK following imposed positive and negative defocus in chickens

Perikles Simon, Marita Feldkaemper, Michaela Bitzer, Sibylle Ohngemach, Frank Schaeffel
 
 

Section of Neurobiology of the Eye, University Eye Hospital, Tuebingen, Calwerstr, 7/1, 72076 Tuebingen, Germany

Correspondence to: Perikles Simon, MD, PhD, Section of Neurobiology of the Eye, University Eye Hospital, Tuebingen, Calwerstr, 7/1, 72076 Tuebingen, Germany; Phone: ++49-7071-2982186; FAX: ++49-7071-294846; email: perikles@uni-tuebingen.de

Abstract

Purpose: Imposing defocus to the retina results in compensatory changes of axial eye growth. It is not clear which factors initially contribute to this process and whether they act on the post-translational, translational, or transcriptional level. We have measured early changes in mRNA levels, in response to imposed negative and positive defocus, of the transcription factor ZENK, the retinoic acid synthesis enzyme RALDH-2, and the growth factor TGFβ-2.

Methods: Chickens 11 days of age were unilaterally treated with positive or negative spectacle lenses of 7 D power. After 0, 15, 30, and 120 min, mRNA was extracted from retina and choroid, and the concentration of the mRNAs of the three candidates was measured by quantitative real time PCR in both eyes.

Results: ZENK in the retina and RALDH-2 in the choroid displayed parallel signs of defocus dependent changes in mRNA levels after 15 or 30 min, respectively. ZENK mRNA levels were reduced in the retina after 15 min with both types of lenses but were then up regulated at 30 min with positive lenses and down regulated with negative lenses, similar to the previously observed changes in ZENK protein levels. Changes of RALDH-2 and TGFβ-2 mRNA levels were confined to the choroid. Treatment with negative lenses resulted in a rapid (15 min) and persistent decrease in TGFβ-2 mRNA concentration in the choroid. Negative lenses provoked parallel but less pronounced alterations in the open fellow eyes.

Conclusions: Imposed defocus triggers extensive transcriptional changes of ZENK in the retina, and of TGFβ-2 and RALHD-2 in the choroid. Changes in retina and choroid are rapid, show no phase delay with respect to each other, and can be considered, in the case of RALDH-2 and ZENK, as specific for the sign of imposed defocus. They occur prior to any morphological changes. This is consistent with a role in causing or controlling later changes in eye growth.

Introduction

If emmetropic animals wear negative or positive spectacle lenses, their eyes respond with enhanced or reduced axial growth, respectively. The growth changes compensate for the imposed refractive errors, largely independent of brain processing or accommodation [1]. Since it is possible that the underlying biochemical mechanisms are similar during refractive error development in humans [2], it is important to understand the biochemical pathways of the feedback loops which appear to reside largely in the fundal layers.

Biochemical substances that are thought to be involved in retinal control of eye growth include the growth factor TGFβ [3-5], retinoic acid [6,7], and the nuclear transcription factor ZENK (also known as Zif269, EGR-1, NGFI-A, and Krox-24). ZENK has recently gained attention because it was the first protein that is regulated in correlation with signs of defocus [8,9]. If hyperopic defocus is imposed by negative lenses (which induces myopic eye growth), the expression of the ZENK protein in glucagonergic amacrine cells is reduced, whereas if myopic defocus is imposed with positive lenses (which induces hyperopic eye growth), ZENK expression is enhanced. Interestingly, the changes in ZENK protein synthesis can be detected as early as 30 min after the beginning of lens wearing, which is clearly ahead of any measurable morphological and biometric changes in the eye. However, ZENK, as a nuclear transcription factor, has no function as a transmitter or growth mediator on its own. Instead, it must exert its effects on ocular growth by altering the expression of tissue remodeling proteins, for example TGFβ, or by altering the levels of retinoic acid.

TGFβ is known to be transcriptionally activated by ZENK via a ZENK binding site in its promotor region [10]. This trans-activation is associated with growth inhibition [10,11]. When axial eye growth is enhanced, both factors are down regulated. For TGFβ, a down regulation of both protein and mRNA content was observed in the retina-RPE-choroid complex following treatment of the chicken with eye occluders for 14 days [5,12]. Furthermore, when myopia was induced, expression of TGFβ was suppressed in the sclera [5]. In vitro studies have shown that TGFβ decreases the proliferation of scleral chondrocytes [4,12], mimicking what happens in vivo when scleral growth is inhibited.

All-trans-retinoic acid (at-RA) is a ligand of the RA receptor which, upon activation, acts as a nuclear transcription factor [13], similar to ZENK. A variety of genes involved in metabolic activity and growth processes have been described that respond to RA receptor activation. At-RA bound to RA receptors trans-activates or transcriptionally suppresses both TGFβ and ZENK expression. The inhibitory or activating potential seems to depend on the presence or absence of cofactors [14]. The concentration of at-RA in choroid and retina is modulated by the kind of visual experience that changes axial eye growth [15]. Similar to ZENK in the retina and TGFβ in retina-RPE-choroid complex, at-RA content is reduced in the choroid during induction of myopia and increased during induction of hyperopia. Moreover, like TGFβ, at-RA inhibits the proliferation of cartilaginous tissue in vitro [7]. Although TGFβ, ZENK, and at-RA all are up regulated when eye growth is inhibited and down regulated when eye growth is enhanced, in vitro experiments on cultured retinal pigment epithelial cells have shown that at-RA can reduce the concentration of both TGFβ mRNA and protein [16].

Here, we further study the interactions of TGFβ, ZENK, and at-RA during the development of refractive errors. Since previous studies on retinoic acid or TGFβ provided data only for time points at which the compensatory growth response of the eye had already saturated, we start our measurements soon after the beginning of the lens treatment. This excludes that the measured changes are secondary and a consequence rather than a cause of altered eye growth. Also we study whether the changes take place first in retina or choroid. Quantitative real time PCR analysis was used to measure early transcriptional changes of ZENK, RALDH-2, which is the predominant source of at-RA [17], and TGFβ-2, the only secreted TGFβ isoform in the retina-choroid complex [18], in both retinal and choroidal tissue [19]. Particular efforts were made to exclude potential pitfalls of the quantitative real time PCR, and the resulting conclusions may be broadly useful to optimize protocols in other studies.

Methods

Treatment of animals and tissue preparation

Twenty-one male white leghorn chickens were raised after hatching under a 12 h light/12 h dark cycle for eleven days. Their treatment was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the institutional review board. Either negative or positive lenses with 7 D power were attached to Velcro rings which were glued to the feathers around the eye in the morning of the day before the experiment. This excludes that any potential changes in mRNA levels were induced by anesthesia, glue, or attachment of the Velcro rings. Only one eye was treated and the contralateral eye was used as control. All animals were sacrificed at the same time on the following day between noon and 30 min past noon, following a treatment period with lenses of 0, 15, 30, and 120 min. Tissue from 3 animals was analyzed for each treatment period.

Animals were killed by decapitation after diethylether anesthesia and eyes were immediately enucleated. The retina, including the adjacent retinal pigment epithelium (RPE), was separated from the choroid and care was taken to avoid cross contamination between tissues. The retina/RPE complex is referred to as "retina" below. The separation of retina with attached RPE from the choroid was facilitated if the preparation of the tissues occurred in ice chilled Ringer solution with a pH of 7.4. Contamination of choroidal tissue with RPE cells was prevented by carefully brushing off small residual RPE cell clusters under visual control through a dissecting microscope. The removed clusters were added to the retinal tissue samples. Choroid and retina were immediately frozen in liquid nitrogen. All tissues were stored at -70 °C for less than a week before total RNA was isolated.

Preparation of RNA and reverse transcription to cDNA

Total RNA was isolated from retinal and choroidal tissues by Trizol Reagent (Invitrogen, Karlsruhe, Germany), according to the manufacturer's instructions. All samples were subjected to treatment with RNAse free DNAse I (Boehringer, Mannheim, Germany) and the integrity of the purified RNA was verified by agarose gel electrophoresis followed by ethidium bromide staining. The OD₂₆₀/OD₂₈₀ nm absorption ratio was greater than 1.95. Consecutively, 1 μg of every RNA sample was reverse transcribed with 100 U of Superscript II RNase H-Reverse (Invitrogen) using 500 ng oligo (dT)₁₅ primer (Boehringer), according to the manufacturer's instructions. The RNA of treated eyes and their respective contralateral control samples were always reverse transcribed in the same run. The variability of the reverse transcription reaction was analyzed by reverse transcribing 6 subsets of the same RNA sample and determining the variability of expression levels of ZENK, TGFβ-2, and RALDH-2 in 5 repetitions of the quantitative real time RT-PCR. Stability of expression levels under experimental and control conditions was investigated for 18S ribosomal RNA (18S RNA) that served as a housekeeping gene in the present study.

Quantitative real time RT-PCR

The sequences of the PCR primers used in this study are listed in Table 1. Primer design was done with the software Primer3 [20] and Primer Premier 5 (PREMIER Biosoft International, Palo Alto, CA). All primers were chosen so that they had close to an equal optimal annealing temperature of 60 °C and a similar GC content between 55.0% and 63.2%. They were synthesized by Interactiva (Thermo Hybaid, Ulm, Germany). Specificity of the primers was verified by determining amplicon sizes by gel electrophoresis and by melting curve analysis. In addition, the specificity of the selected primers was verified by a Blast search of the GenBank data base. We were careful to ensure that no other closely related sequences of the retinaldehyde family or the TGFβ family matched the selected primers.

PCR was performed using standard protocols with SYBR®Green as a fluorescent detection dye in a real time iCycler from Biorad (Hercules, CA). All PCR reactions had a final volume of 15 μl, comprised of SYBR Green PCR kit, 400 μM forward and reverse primer, and 1 ng of reverse transcribed RNA. The reaction was pipetted on ice into a 96 well plate (Abgene, Rochester, NY). The plate was heat sealed with optical quality sealing tape (BioRad, Hercules, CA), and the well contents were collected by brief centrifugation of the plate.

For gene specific amplification, we used the following PCR cycle parameters: Polymerase activation for 15 min at 95 °C and 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 60 s. Fluorescence was measured at 72 °C. Melting curve analysis between 50 °C and 100 °C at 0.5 °C intervals was finally applied to characterize the generated amplicons and to control for contamination by nonspecific byproducts.

Standard curves for six 2 fold dilution steps between 4 ng and 0.125 ng of reverse transcribed RNA samples were run for all primer pairs in triplicate to determine the PCR efficiency for the different target genes and the housekeeping gene. All PCR reactions for a given sample were pipetted in 5 replicates to control for the variability of the PCR amplification. After the exclusion of outliers, as determined by irregularities in their amplification curves or their melting curves, the averages of the replicates were used for statistical data processing. A total of 2,100 PCR reactions was performed in this study but only 28 PCRs were excluded because of such irregularities. Therefore, 396 quintet, 24 quadruple, and 2 triple repetitions were analyzed.

Real time PCR data analysis and statistics

For all PCR run evaluations, "Cycle Threshold" values (CT) were calculated for the different products. The CT value is defined as the PCR cycle where the fluorescence intensity of an amplicon crosses a threshold line. The threshold was set in the range of exponential amplification at 1/10 of the average maximum fluorescence. The CT obtained for the different amplicons were statistically processed by the software package REST, applying a pair wise fixed reallocation randomization test (REST), which has been designed especially for evaluation of semi-quantitative real time PCR data [21]. This evaluation procedure takes the different amplification efficiencies for the target genes (E_target) and the reference gene (E_reference) into consideration and determines whether the expression of a target gene relative to a reference gene is significantly different between an experimental group and a control group. Additionally, REST is well suited for statistical comparisons of both normal and non-normal distributions [22].

For the statistical comparison of the average expression level in contralateral control and lens treated eyes after 0, 15, 30, and 120 min (n=3 at every time point), separate ANOVAs for the two different treatment conditions (+7 D and -7 D lenses) were performed. In the analysis of the time courses, only three eyes were entered at the zero minute point for the ANOVA testing for both the positive and negative lens treated groups. Since, in some cases, homogeneity of variance tests indicated that the variances across groups were variable, Welch ANOVA had to be used instead of the standard ANOVA F test. Only significant ANOVAs were followed by REST as a post hoc analysis. For the comparison of the differences in gene expression at a given time between treated and control eyes, a Bonferroni corrected paired Student's t test was added. P values of <0.017 (for the three pair-wise comparisons) were considered significant.

For the graphical presentation of the results and statistical analyses by paired t tests and ANOVA, Mean Normalized Expressions (MNEs) of the target genes were calculated. MNEs are linear expression values of a target gene relative to a reference gene, calculated from the average CT value of the five replicates for the target (CT_{target, mean}) and for the appropriate reference gene (CT_{reference, mean}). Despite the fact that the MNEs were calculated from the logarithmically scaled raw data, they offer the possibility to calculate standard errors for the MNE, which reflect the variation of the raw data [23].

Results

Specificity and variability of the semi-quantitative real time PCR

Melting curves displayed a single product at the specific melting temperature (Figure 1A) which was calculated by the nearest neighbor procedure [24]. Within a range of 0.5 °C, the measured melting temperatures for 18S RNA (84.3 °C), ZENK (82.0 °C), RALDH-2 (82.8 °C), and TGFβ-2 (81.9 °C) matched their theoretical values [25]. In addition, gel electrophoresis of RT-PCR products revealed single bands for the four different primer pairs with the desired length (Figure 1B). Although the PCR products were not subjected to DNA sequence analysis, they were the exact sizes predicted. Also, because product formation was dependent on both primers being present in PCR, it is highly likely that they are authentic.

To further verify the specificity of the primers, PCR was performed with tissue of mouse forebrain and mouse retina. The amounts of cDNA were identical to the ones used in the experiments with chicken retina and choroid. The primers for ZENK, RALDH-2, and TGFβ-2 caused no amplification of transcripts or at least displayed CT values that were more than 6 cycles higher than the highest values found in the experiments with chicken tissue. No primer-dimers or nonspecific byproducts were generated during 40 real time PCR amplification cycles.

To verify the reproducibility of the real time PCR, the intra-assay variability was analyzed in 5 repetitions for each cDNA sample, amplified with four different primer pairs. The average coefficients of variation (CV) in 84 measurement series are illustrated in Figure 2A for the CT values of the four transcripts. They were as follows; 18S RNA (1.2%), ZENK (0.9%), RALDH-2 (1.2%), and TGFβ-2 (1.1%).

To compare gene expression levels of the three different target genes within different samples, the raw data CT values were processed by calculating the Mean Normalized Expression values (MNEs), which describe the expression of a target gene relative to a control gene. MNEs allow comparisons between target genes since they take the different amplification efficiencies and the level of the housekeeping gene into account. The variability of the data from the real time PCR is therefore correctly represented by the CV of the MNE (Figure 2B), more realistically than by the CV of the raw data, which have considerably lower values (Figure 2A).

Consistency of the reverse transcription step was controlled by reverse transcribing six subsets of the same RNA probe and determining the CT for the target genes by real time PCR. Five repetitions for each cDNA probe were performed. Figure 3A illustrates the small variability of the average CT, based on the different cDNA samples (CV ranging from 0.4% to 0.8%).

Amplification efficiency and stability of housekeeping gene expression

For the evaluation of the amplification efficiencies during PCR, dilution series were run and standard curves were generated for all the primer pairs (Figure 3B). The correlation coefficients suggested a high linearity for the different targets within the tested range of 4.0-0.125 ng (Pearson correlation coefficient: r=0.98 for the three target genes and 0.99 for the housekeeping gene). The corresponding amplification efficiencies (E) were calculated according to the standard equation; E=(-10/slope)-1. The transcripts under consideration showed high and homogenous real time PCR efficiencies, with E_{18S RNA}=0.97, E_ZENK=1.03, E_RALDH-2=0.99; and E_TGFβ-2=0.98. The stability of the housekeeping gene during treatment with positive (Figure 3C) and negative lenses (Figure 3D) was analyzed in nine animals. Neither -7 D nor +7 D lenses caused a statistically significant change in expression of 18S RNA in retina or choroid.

Defocus imposed by -7 D and +7 D lenses alters the concentration of ZENK mRNA in the retina

Of the three investigated transcripts, only ZENK mRNA concentrations were significantly changed following treatment with lenses. In the eyes treated with -7 D lenses, ZENK mRNA concentrations decreased within 30 min by 67%, compared to the level at 0 min (Figure 4, p<0.005, REST). The decrease was even more pronounced after 120 min of treatment (92%) and, at this point, it was accompanied by a significant decrease of 82.0% in the contralateral control eye (p<0.005 for both eyes, REST).

In contrast, following treatment with positive lenses ZENK mRNA concentration was not significantly altered in the control eyes. A statistically significant decrease (52%) in ZENK expression was observed in the lens treated eyes 15 min after the beginning of lens treatment (compared to the contralateral control eye; p<0.05, paired t test). After 30 min, the level of ZENK mRNA was up regulated. Compared to the baseline level, ZENK mRNA concentration was increased by 32%. The increase was significant compared to that in the contralateral control eye (+28%, p<0.05, paired t test). Neither retinal RALDH-2 nor TGFβ-2 mRNA concentrations were significantly changed by the lens treatment. In the case of RALDH-2, the high CT values of the raw data (on average about 32) added to the high variability and rendered the differences nonsignificant, as indicated by Welch ANOVA testing.

Levels of TGFβ-2 mRNA showed a high pre-treatment variability in the MNE (range from 52x10^-5 to 333x10^-5). Treatment with negative lenses tended to reduce RALDH-2 expression after 30 and 120 min in both treated and control eyes by about 50%. With positive lenses, an increase of 70% was observed but, as stated above, these changes did not achieve significance because of the large baseline variability.

Changes in choroidal mRNA levels following defocus

The levels of mRNA in retina and choroid were clearly different (Figure 5). While the average MNE of ZENK was 5 fold lower in the choroid than in the retina (101x10^-5 versus 22x10^-5), the expression level of RALDH-2 was 15 times higher in the choroid than in the retina (77x10^-5 versus 5.4x10^-5). Therefore, quantification of RALDH-2 mRNA was more reliable in the choroid with average CT values around 28 than in the retina with average CT values close to 32 (Figure 3A). On the other hand, both tissues showed quite similar MNEs for TGFβ-2, with 165x10^-5 in the retina and 171x10^-5 in the choroid.

Treatment with negative lenses reduced the mRNA concentrations of TGFβ-2 in the choroid rapidly and consistently. At 15 min, the MNE was decreased by 81% (p<0.01, REST) and remained at the low level after 30 min and 120 min of negative lens wear (30 min: 76%, p<0.01; 120 min: 93%, p<0.005, REST). The decrease was accompanied by a statistically significant decrease in the contralateral control eyes, at both 15 and 30 min (15 min: 71%, p<0.05; 30 min 70%, p<0.05; and 93% at 120 min, p<0.005, REST). Although the effects were small, the mean mRNA concentrations of TGFβ-2 were significantly lower in the eyes treated with negative lenses, compared to the contralateral eyes (at 15 and 30 min: p<0.01, by two tailed paired t tests).

Choroidal ZENK and RALDH-2 mRNA concentrations were down regulated in control and lens treated eyes after 120 min of negative lens treatment (p<0.005, REST). At both 15 min and 30 min, RALDH-2 mRNA concentrations were already significantly lower than in the untreated control group (-49% and -53% in the treated eyes, respectively, p<0.05 for both, REST). However, no significant differences were observed between the contralateral control eyes and treated eyes for RALDH-2 at any time. This was different for ZENK where the decline of mRNA concentration was more pronounced in the treated than in the open eyes after 15 min (25% versus 50%, p<0.05, REST).

With positive lenses, the only significant change was an increase in RALDH-2 expression at 15 min (+64% compared to the contralateral eye, p<0.01 by a two tailed t test). At this time, the expression of RALDH-2 in the treated eye was increased by 244%, compared to baseline at zero minutes. Therefore, the RALDH-2 mRNA level was the only of the three investigated mRNAs that was regulated in parallel with the sign of defocus after 15 min, both in retina and choroid.

Comparison of mRNA concentrations and protein concentrations for ZENK

The present data on the concentration of the ZENK mRNA and can be compared to previous data from immunohistochemical studies [9] to uncover possible correlations between mRNA content and protein content. With normal visual experience, about 50% of the glucagonergic cells in the chicken retina are co-expressing the ZENK protein. Following treatment with negative lenses, the mRNA, as well as the ZENK protein concentrations, dropped severely after 120 min (Figure 6A). Interestingly, ZENK protein expression increased considerably (+56%) also in the contralateral eye after 30 min, compared to the level at zero minutes. Similar to the mRNA, the increase was followed by a decline at 120 min which was similar in both eyes. The major difference between the mRNA and the protein concentration was, therefore, the time course in the first 30 min of lens treatment.

A similar result was observed with positive lenses (Figure 6B). Despite the initial decline of mRNA concentration in both treated and control eyes after 15 min (with even lower concentration in the treated eye; p<0.05, paired t test), the protein content and relative mRNA level were increasing after 30 min, again in both the contralateral and the lens treated eye (protein and mRNA: p<0.05, paired t test).

Discussion

We have found changes in mRNA levels of ZENK, TGFβ-2, and RALDH-2 in retina and choroid following imposition of myopic or hyperopic defocus, which occur clearly before any morphological changes are detectable. These changes are the earliest responses to imposed defocus that were detected so far, and they are, in part, even specific for the sign of defocus. The most surprising findings were the changes in RALDH-2 in the choroid, which were parallel to the sign of imposed defocus after only 15 min. Also, other findings were unexpected: ZENK mRNA concentrations in the retina initially dropped with both types of lenses, and there was extensive co-regulation of mRNA levels in the open fellow eyes, even though previous studies have shown that they are not followed by changes in eye growth.

Reliability of real time PCR measurements

Quantitative real time PCR was found to be a powerful technique to measure relative mRNA levels of candidate genes. The coefficient of variation of data from the quantitative real time PCR varies depending on the transcript investigated between 25% and 31%. It was markedly different for raw data (CTs) or normalized expressions (range for the raw data was 0.9% to 1.2%). Raw data of quantitative real time PCR are logarithmic and do not reflect relative expression levels. The variability is better reflected in the CV_MNEs, as shown in Figure 2B. Our studies provide reference values for the variability of simplex quantitative real time PCR relative expression data. Recently, one of the authors showed that standard procedures may provide inappropriately low estimates for the variability [23]. However, compared to conventional mRNA quantification strategies such as probe hybridization and band densitometry [26], the variabilities described here are still about 20% lower. This confirms the high sensitivity of the real time PCR technique to quantify differences in mRNA levels.

Correlations of ZENK mRNA levels and ZENK protein expression

Changes in ZENK mRNA concentration in the retina were surprisingly well correlated with the previously reported [9] changes in ZENK protein concentration (Figure 6) although the correlation was not preserved at all time points. For instance, 30 min of negative lens wear reduced mRNA levels, whereas the number of ZENK immunoreactive cells was still stable in the treated eye and was even increasing in the contralateral eye. This differential regulation of mRNA and protein, however, could be due to a reinforced translation of the resident mRNAs to generate the protein as demonstrated, for instance, in neurons for calcium calmodulin kinase IIa [27]. This assumption would also explain the initial decrease of ZENK mRNA following imposed positive defocus. Later, after 30 min, mRNA levels were up regulated, which indicates an increased demand.

Changes in RALDH-2 mRNA

With normal visual experience, mRNA levels of RALDH-2, measured as mean normalized expression (MNE_RALDH-2), were 15 times higher in the choroid than in the retina. A similar difference between these two tissues has already been described for the concentration of at-RA as the product of RALDH-2, which was 8.5 fold higher in the choroid than in the retina [15]. RALDH-2 mRNA concentration was found to decrease in the choroid following treatment with negative lenses and to increase with positive lenses. These data are complementary to the measurements of at-RA levels after long term defocus [15]. That mRNA concentration of the enzyme RALDH-2 and the concentrations of at-RA as its product are correlated is not trivial, given that enzymatic activity is often regulated by post-translational modifications, leading to reduced or increased enzyme activity, and not by actual changes in the enzymes concentration or its mRNA levels. However, in this case, mRNA levels of RALDH-2 may reflect changes in the at-RA concentration, especially since RALDH-2 is the prominent source of at-RA [28]. This is of interest because measurements of at-RA levels require large amounts of tissue but quantitative real time PCR can easily be done with as little as 5 ng of total RNA, which can be obtained from less than 1,000 cells.

Alterations of RALDH-2 and TGFβ-2 mRNA concentrations

Another surprising result was that levels of both RALDH-2 and TGFβ-2 mRNA remained largely unchanged during lens treatment in the retina, but changed rapidly in the choroid. Our results are consistent with the results of an earlier study [12], which reported decreased levels of TGFβ-2 mRNA in the retina-RPE-choroid complex in myopic animals. Moreover, we found a drop in TGFβ-2 gene expression in the choroid after treatment with negative lenses for only 15 min, which is before morphological changes are detectable. RALDH-2 gene expression also changed in 15 min, both with negative and positive lenses. These observations show that the information on the sign of defocus may be transmitted to the choroid in only a few minutes.

TGFβ-2 has been suggested as a mediator between retina and choroid, since it is known to be secreted by the RPE [19]. The stability of MNEs for TGFβ-2 in retina, compared to those in choroid, challenges this assumption. Our results suggest that yet another factor might carry the retinal growth signal to the choroid. Previous studies [29,30] have shown that glucagon, a peptide hormone that is co-expressed with ZENK in the subset of amacrine cells that are sensitive to the sign of defocus [8], might be a promising candidate.

The question of the "contralateral control eye"

Because measuring mRNA levels in individual animals is time consuming and expensive, many studies on transcription levels use tissue samples pooled from several animals. However, our measurements show that certain transcripts, like TGFβ-2, vary considerably among individuals (by as much as a factor of 6) even at resting conditions. The variability between two eyes of the same individual may be substantially lower. Therefore, variability may be reduced by paired comparisons between differently-treated eyes of the same animal, an advantage that is lost when tissue from different animals is pooled. In the case of TGFβ-2, the maximal difference between both eyes was only a factor of 2.2, compared to the inter-individual factor of 6 above. Changes in both eyes may be linked despite asymmetrical treatments. This was often observed in the present study. A number of previous studies demonstrated contralateral effects even though the final changes in eye growth were confined to the treated eye [9,31]. Figure 6 shows that changes in the contralateral control eyes occur on the level of the ZENK protein as well as on the level of its mRNA. The pathways connecting both eyes remain obscure. Both systemic humoral factors and neuronal coupling may be involved. In the developing chicken, there exists a direct connection via retino-retinal projections of retinal ganglion cell axons [32].

Conclusions

Imposed defocus, which produces directional changes in eye growth, is accompanied by extensive transcriptional changes in retina and choroid after only 15 min. This places these processes in a similar time range as contrast adaptation, which is based on synaptic plasticity [33] and learning [34]. The changes in the choroid show no time delay with respect to the changes in the retina, suggesting that signals are transmitted from the retina to the choroid with little or no delay. The clearest sign of defocus dependence is displayed by ZENK mRNA levels in the retina and RALDH-2 mRNA levels in the choroid. Even though the final growth changes have been shown to be largely independent in both eyes, all the early transcriptional changes measured in this study show extensive co-regulation in both eyes.

Acknowledgements

We thank Dr. Hans-Peter Wendel and his research group at the Department of Thoracic, Cardiac and Vascular Surgery for generously providing working time on technical equipment. We especially thank Dr. Eva Tolosa for an introduction into real-time RT-PCR and for her indispensable help in discussing and solving technical problems. This study has been supported in part by the German Research Council, SFB 430, TP C1.

References

1. Wallman J. Retinal control of eye growth and refraction. Progress in Retinal Research 1993; 12:134-53.

2. Wallman J. Nature and nurture of myopia. Nature 1994; 371:201-2.

3. Rohrer B, Stell WK. Basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF-beta) act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res 1994; 58:553-61.

4. Seko Y, Tanaka Y, Tokoro T. Influence of bFGF as a potent growth stimulator and TGF-beta as a growth regulator on scleral chondrocytes and scleral fibroblasts in vitro. Ophthalmic Res 1995; 27:144-52.

5. Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-beta 2 in experimental myopia in chicks. Invest Ophthalmol Vis Sci 1995; 36:1183-7.

6. Seko Y, Shimizu M, Tokoro T. Retinoic acid increases in the retina of the chick with form deprivation myopia. Ophthalmic Res 1998; 30:361-7.

7. Seko Y, Shimokawa H, Tokoro T. In vivo and in vitro association of retinoic acid with form-deprivation myopia in the chick. Exp Eye Res 1996; 63:443-52.

8. Fischer AJ, McGuire JJ, Schaeffel F, Stell WK. Light- and focus-dependent expression of the transcription factor ZENK in the chick retina. Nat Neurosci 1999; 2:706-12.

9. Bitzer M, Schaeffel F. Defocus-induced changes in ZENK expression in the chicken retina. Invest Ophthalmol Vis Sci 2002; 43:246-52.

10. Liu C, Adamson E, Mercola D. Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta 1. Proc Natl Acad Sci U S A 1996; 93:11831-6.

11. Liu C, Rangnekar VM, Adamson E, Mercola D. Suppression of growth and transformation and induction of apoptosis by EGR-1. Cancer Gene Ther 1998; 5:3-28.

12. Honda S, Fujii S, Sekiya Y, Yamamoto M. Retinal control on the axial length mediated by transforming growth factor-beta in chick eye. Invest Ophthalmol Vis Sci 1996; 37:2519-26.

13. Kurlandsky SB, Xiao JH, Duell EA, Voorhees JJ, Fisher GJ. Biological activity of all-trans retinol requires metabolic conversion to all-trans retinoic acid and is mediated through activation of nuclear retinoid receptors in human keratinocytes. J Biol Chem 1994; 269:32821-7.

14. Lefebvre P. Molecular basis for designing selective modulators of retinoic acid receptor transcriptional activities. Curr Drug Targets Immune Endocr Metabol Disord 2001; 1:153-64.

15. Mertz JR, Wallman J. Choroidal retinoic acid synthesis: a possible mediator between refractive error and compensatory eye growth. Exp Eye Res 2000; 70:519-27.

16. MacDonald IM, Pannu R, Kovithavongs K, Peters C, Tredget EE, Ghahary A. Effect of retinoic acid on expression of transforming growth factor-beta by retinal pigment epithelial cells in culture. Can J Ophthalmol 1995; 30:301-5.

17. Niederreither K, McCaffery P, Drager UC, Chambon P, Dolle P. Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development. Mech Dev 1997; 62:67-78.

18. Tanihara H, Yoshida M, Matsumoto M, Yoshimura N. Identification of transforming growth factor-beta expressed in cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 1993; 34:413-9.

19. Pfeffer BA, Flanders KC, Guerin CJ, Danielpour D, Anderson DH. Transforming growth factor beta 2 is the predominant isoform in the neural retina, retinal pigment epithelium-choroid and vitreous of the monkey eye. Exp Eye Res 1994; 59:323-33.

20. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 2000; 132:365-86.

21. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002; 30:e36.

22. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29:e45.

23. Simon P. Q-Gene: processing quantitative real-time RT-PCR data. Bioinformatics 2003; 19:1439-40.

24. Breslauer KJ, Frank R, Blocker H, Marky LA. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A 1986; 83:3746-50.

25. Sugimoto N, Nakano S, Yoneyama M, Honda K. Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res 1996; 24:4501-5. Erratum in: Nucleic Acids Res 2001; 29:2.

26. Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem 2000; 285:194-204.

27. Wu L, Wells D, Tay J, Mendis D, Abbott MA, Barnitt A, Quinlan E, Heynen A, Fallon JR, Richter JD. CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses. Neuron 1998; 21:1129-39.

28. Niederreither K, Subbarayan V, Dolle P, Chambon P. Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. Nat Genet 1999; 21:444-8.

29. Buck C, Schaeffel F, Simon P, Feldkaemper M. Effects of positive and negative lens treatment on retinal and choroidal glucagon and glucagon receptor mRNA levels in the chicken. Invest Ophthalmol Vis Sci 2004; 45:402-9.

30. Feldkaemper MP, Schaeffel F. Evidence for a potential role of glucagon during eye growth regulation in chicks. Vis Neurosci 2002; 19:755-66.

31. Siegwart JT Jr, Norton TT. The time course of changes in mRNA levels in tree shrew sclera during induced myopia and recovery. Invest Ophthalmol Vis Sci 2002; 43:2067-75.

32. Thanos S. Genesis, neurotrophin responsiveness, and apoptosis of a pronounced direct connection between the two eyes of the chick embryo: a natural error or a meaningful developmental event? J Neurosci 1999; 19:3900-17.

33. Diether S, Gekeler F, Schaeffel F. Changes in contrast sensitivity induced by defocus and their possible relations to emmetropization in the chicken. Invest Ophthalmol Vis Sci 2001; 42:3072-9.

34. Wei F, Xu ZC, Qu Z, Milbrandt J, Zhuo M. Role of EGR1 in hippocampal synaptic enhancement induced by tetanic stimulation and amputation. J Cell Biol 2000; 149:1325-34.