Alterations in ZENK and glucagon RNA transcript expression during increased ocular growth in chickens

Abstract

Purpose: To examine in detail the time-course of changes in Zif268, Egr-1, NGFI-A, and Krox-24 (ZENK) and pre-proglucagon (PPG) RNA transcript levels in the chick retina during periods of increased ocular growth induced by form-deprivation and negative-lens wear. To further elucidate the role of ZENK in the modulation of ocular growth, we investigated the effect of intravitreal injections of the muscarinic antagonist atropine and the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (ADTN), both of which block the development of experimental myopia, on the expression of ZENK in eyes fitted with negative-lenses.

Methods: Myopia was induced by fitting translucent diffusers or -10D polymethyl methacrylate (PMMA) lenses over one eye of the chicken. At times from 1 h to 10 days after fitting of the diffusers or negative lenses, retinal RNA transcript levels of the selected genes were determined by semi-quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). For the pharmacology experiments, -10D lenses were fitted over the left eye of chicks for a period of 1h. Intravitreal injections of atropine (10 mul-25 mM), ADTN (10 mul-10 mM), or a vehicle solution were made immediately before fitting of the lenses.

Results: ZENK RNA transcript levels were rapidly and persistently down-regulated following the attachment of the optical devices over the eye. With a delay relative to ZENK, PPG transcript levels were also down-regulated. Induced changes in gene expression were similar for both form-deprivation and negative-lens wear. When atropine or ADTN were administered immediately before lens attachment, the rapid down-regulation in ZENK RNA transcript levels normally seen following 1 h of negative-lens wear was not seen, and ZENK transcript levels rose above those values seen in control eyes. However, injection of atropine or ADTN into untreated eyes had no effect on ZENK transcript levels.

Conclusions: Both form-deprivation and negative-lens wear modulated the retinal expression of ZENK and PPG RNA transcripts, with a similar time-course and strength of response. The ability of the tested drugs to prevent the down-regulation of ZENK in both lens-induced myopia (LIM) and form-deprivation myopia (FDM) suggests that atropine and ADTN act directly and rapidly on retinal circuits to enhance sensitivity early in the signaling process. These findings suggest that very similar molecular pathways are involved in the changes in eye growth in response to form-deprivation and negative lenses at 1 h after the fitting of optical devices.

Figure 1

Changes in the refractive error of treated and contralateral control eyes over ten days of form-deprivation and negative-lens wear. The fitment of translucent diffusers over the eye induced significant development of myopia over the ten-day experimental period, as compared to control values (MANOVA; F (1,10)=8.3, p<0.01). Chicks fitted with −10D lenses significantly compensated for the lenses over the initial seven days of treatment (MANOVA; F (1,7)=10.2, p<0.01), before plateauing. Although changes in refraction of contralateral control eyes appeared different between treatment groups, this behavior was not statistically significant over time (MANOVA; F (1,10)=2.04, p=0.18). Error bars represent the standard error of the mean (SEM), n=8 per time, per experimental treatment.

Figure 2

Changes in ZENK RNA transcript levels in treated and contralateral control retinas following increased ocular growth induced by the fitting of translucent diffusers or negative lenses. Mean normalized expression of ZENK RNA transcript levels from diffuser-treated (A) or negative-lens-treated (B) eyes following 1 h, 1, 3, 7, and 10 days of treatment. Fitting of translucent diffusers or negative lenses significantly affected ZENK RNA transcript levels in the experimental eye over time, as compared to both contralateral control values (MANOVA; F (2,86)=33.9, p<0.01 and F (2,86)=32.9, p<0.001, respectively) and age-matched untreated control values (MANOVA; F (2,86)=37.9, p<0.001 and F (2,86)=18.5, p<0.01, respectively). ZENK transcript levels in the contralateral control eyes from either form-deprived or negative-lens-treated animals were unaffected by treatment as compared to age-matched untreated values (MANOVA; F (2,86)=2.65, p=0.14 and F (2,86)=0.69, p=0.43, respectively). The mean normalized expression is calculated from the efficiency (E) of the target genes to the power of its average CT value (E^CT, target), divided by the efficiency (E) of the reference gene (β-actin) to the power of its average CT value (E^CT, reference). Error bars represent SEM, n=9. (* p<0.05, ** p<0.01).

Figure 3

Changes in ZENK RNA transcript levels in the chick retina over a 24 h time period. ZENK transcript levels in the retina were significantly suppressed following 1 h of form-deprivation, and remained suppressed during the subsequent dark phase and the beginning of the following light phase, as compared to age-matched control values (ANOVA; F (2,46)=23.45, p<0.001) and contralateral control values (ANOVA; F (2,46)=13.34, p<0.01). The mean normalized expression is calculated from the efficiency (E) of the target genes to the power of its average CT value (E^CT, target), divided by the efficiency (E) of the reference gene (β-actin) to the power of its average CT value (E^CT, reference). Error bars represent SEM, n=5.

Figure 4

Changes in pre-proglucagon RNA transcript levels in treated and contralateral control retinas during periods of increased ocular growth induced by the fitment of translucent diffusers or negative lenses. Mean normalized expression of PPG RNA transcript levels from diffuser-treated (A) and negative-lens treated (B) eyes following 1 h, 1, 3, 7, and 10 days of treatment. The fitting of translucent diffusers or negative lenses significantly affected PPG RNA transcript levels in the experimental eye over time, as compared to both contralateral control values (MANOVA; F (2,86)=13.29, p<0.05 and F (2,86)=8.31, p<0.05, respectively) and age-matched untreated control values (MANOVA; F (2,86)=13.02, p<0.05 and F (2,86)=9.12, p<0.05, respectively). There was no significant difference in the expression of PPG transcript levels over time, between contralateral control eyes from either form-deprived animals or negative-lens-treated animals and age-matched untreated values (MANOVA; F (2,86)=0.27, p=0.62 and F (2,86)=0.10, p=0.84, respectively). The mean normalized expression is calculated from the efficiency (E) of the target genes to the power of its average CT value (E^CT target), divided by the efficiency (E) of the reference gene (β-actin) to the power of its average CT value (E^CT reference). Error bars represent SEM, n=9. (* p<0.05, ** p<0.01).

Figure 5

The effect of atropine and ADTN on ZENK transcript levels in the retina following 1 h of negative-lens wear. Negative-lens wear, for a period of 1h, induced significant down-regulation in ZENK transcript levels (ANOVA; F (4, 20)=4.24, p<0.05; t-test, p<0.05, respectively), as compared to normal untreated values, which was unaffected by the injection of either vehicle solution (distilled waster or ascorbic acid) immediately before lens fitting (t-test; p=0.7 and p=0.8, respectively). However, injection of atropine or ADTN immediately before the attachment of lenses induced significant upregulation in retinal ZENK expression above baseline levels (ANOVA; F (3, 14)=6.32, p<0.05; t-test, p<0.05, respectively). Atropine or ADTN did not affect retinal ZENK expression when injected into a normal untreated age-matched eye (ANOVA; F (3,14)=0.78, p=0.11; t-test, p=0.15, p=0.10, respectively). The mean normalized expression is calculated from the efficiency (E) of the target genes to the power of its average CT value (E^CT target), divided by the efficiency (E) of the reference gene (Actb) to the power of its average CT value (E^CT reference). Error bars represent SEM, n=6 (*p<0.05).