P. Megaw

Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse

BackgroundNocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse.ResultscDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver.ConclusionThe widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector.

Diurnal patterns of dopamine release in chicken retina

The retinal dopaminergic system appears to play a major role in the regulation of global retinal processes related to light adaptation. Although most reports agree that dopamine release is stimulated by light, some retinal functions that are mediated by dopamine exhibit circadian patterns of activity, suggesting that dopamine release may be controlled by a circadian oscillator as well as by light. Using the accumulation of the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) in the vitreous as a measure of dopamine release rates, we have investigated the balance between circadian- and light control over dopamine release. In chickens held under diurnal light:dark conditions, vitreal levels of DOPAC showed daily oscillations with the steady-state levels increasing nine-fold during the light phase. Kinetic analysis of this data indicates that apparent dopamine release rates increased almost four-fold at the onset of light and then remained continuously elevated throughout the 12h light phase. In constant darkness, vitreal levels of DOPAC displayed circadian oscillations, with an almost two-fold increase in dopamine release rates coinciding with subjective dawn/early morning. This circadian rise in vitreal DOPAC could be blocked by intravitreal administration of melatonin (10 nmol), as predicted by the model of the dark-light switch where a circadian fall in melatonin would relieve dopamine release of inhibition and thus be responsible for the slight circadian increase in dopamine release. The increase in vitreal DOPAC in response to light, however, was only partially suppressed by melatonin. The activity of the dopaminergic amacrine cell in the chicken retina thus appears to be dominated by light-activated input.

Screening for differential gene expression during the development of form-deprivation myopia in the chicken

Purpose. To use the technique of differential gene display to analyze changes in gene expression that occur during the development of and recovery from form-deprivation myopia. Methods. The differential display-reverse transcriptase-polymerase chain reaction technique was used to detect cDNAs that are differentially expressed after 24 h (including 12 h in the light) after fitting with a diffuser to induce form-deprivation myopia. Messenger RNA levels were determined by quantitative Northern blotting in retinas after 11 days of form deprivation or in retinas where the diffusers had been removed the previous day. Results. Twenty-six differentially expressed genes were processed in our initial screen. Two of these, &agr;B-crystallin and retinoic acid receptor-&agr;, were studied further. Levels of &agr;B-crystallin mRNA were increased on day 11 in retinas from form-deprived eyes relative to eyes of control chickens and were reduced to below those levels within 6 to 12 h after removal of the diffusers. Levels of retinoic acid receptor-&agr; mRNA showed similar changes, except that after removal of the diffusers, the levels further increased. Conclusions. The technique of differential gene display can be used to detect changes in gene expression during the regulation of eye growth. The response of &agr;B-crystallin is particularly interesting because expression increases when eye growth is high and decreases when eye growth slows.

Changes in retinal αB-crystallin (cryab) RNA transcript levels during periods of altered ocular growth in chickens

Changes in retinal crystallin gene expression have been implicated in the development of myopia in animal models. We therefore investigated the expression of alphaB-crystallin (cryab) in the chicken retina during periods of increased ocular growth induced by form-deprivation and negative lens-wear, and during periods of decreased ocular growth induced by diffuser removal from previously form-deprived eyes, and plus lens-wear. Cryab RNA transcript levels in the chicken retina were measured using semi-quantitative real-time RT-PCR, at times between 1 h and 10 days after the fitting of diffusers or negative lenses, and at times between 1 h and 3 days following the removal of diffusers from previously form-deprived eyes, or the addition of plus lenses. Changes in expression for each condition at each time-point are analysed relative to expression in retinas from age-matched untreated control birds. No change in relative expression of cryab RNA transcript was detected 1 h after fitting diffusers to induce form-deprivation myopia. A transient increase in cryab RNA transcript expression was detected around 1 day later (p = 0.02), but expression returned to control levels after three days. After 7 (p = 0.005) and 10 (p = 0.001) days, retinal cryab RNA transcript expression progressively increased relative to controls. After removal of the diffusers, to initiate recovery, cryab RNA transcript expression remained elevated, with only a slight return to control levels. During the development of lens-induced myopia, no changes in cryab RNA transcript expression relative to controls were seen on day 1, but increases were seen at 10 days (p = 0.004). No significant changes in retinal cryab RNA transcript expression were seen in response to plus lenses compared to either contralateral control values (MANOVA; F = 0.60, p = 0.48) or age-matched untreated values (MANOVA; F = 4.10, p = 0.08). Changes in retinal cryab RNA transcript expression were not systematically related to changes in the rate of eye growth. The role of the transient increase in cryab expression observed after 1 day of form-deprivation, which was not seen after fitting negative lenses, is unclear. The later increases in relative cryab expression seen during the development of form-deprivation and lens-induced myopia occur too late to have a major role in the differential regulation of eye growth between experimental and control eyes. Given that cryab is a member of the small heat shock protein family, the later increases may reflect the emergence of cell damage related to high myopic pathology in the experimentally enlarged eyes and retina.

Changes in the expression of Pax6 RNA transcripts in the retina during periods of altered ocular growth in chickens.

Genome-wide mapping studies have suggested a possible role for Pax6 in the development of myopia. We therefore investigated the expression of Pax6 RNA transcripts in the chicken retina during periods of increased ocular growth, induced by form-deprivation and negative lens-wear, and during periods of decreased ocular growth, induced by diffuser removal from previously form-deprived eyes, and plus lens-wear. Levels of Pax6 RNA transcripts in the chicken retina were measured using semi-quantitative real-time RT-PCR, at times between 1 h and 10 days after the fitting of diffusers or negative lenses, and at times between 1h and 3 days following the removal of diffusers from previously form-deprived eyes, or the addition of plus lenses. Pax6 expression was unaffected during the initial 3 days of the response to form-deprivation or negative lens-wear, when rapid rates of growth are well-established. Alterations in the expression of Pax6 RNA transcripts were only observed after 7-10 days of form-deprivation (7 days, -15.7 +/- 5.3%; 10 days, -32.0 +/- 10.3%), with a similar response not seen during negative lens-wear, when eye growth also increases, suggesting that these alterations are specific to form-deprivation, rather to changes in the rate of eye growth. The late changes in Pax6 expression observed during form-deprivation were rapidly reversed after diffuser removal, with the levels of Pax6 RNA transcripts returning to those seen in control birds by 3 days (1 h, -27.8 +/- 4.7%; 1 day, -16.9 +/- 4.8%; 3 days + 1.0 +/- 8.6%). Analogous changes were not seen in response to positive lenses in which eye growth is also slowed. Overall, the findings of this study do not support a role for Pax6 in the modulation of ocular growth during visual manipulation.

Light-Driven Rhythms in Scleral Precursor Synthesis

Because there is a diurnal rhythm in axial elongation, and both axial elongation and scleral precursor synthesis increase in form-derivation myopia (FDM), it was expected that there would be a diurnal rhythm in scleral precursor synthesis, with higher values during the day as in axial elongation. However, we have shown that the opposite is in fact true. This rhythm could not be maintained in constant dark conditions, suggesting that it is not under circadian control. The rise does not happen until abut 4h after the lights go off (at 6 p.m.), and there is a gradual decrease over about 6h after the lights come on (at 6 a.m.). Putting the lights on at midnight could also gradually bring the rate down over 6h. This delay suggests that messages may be coming from the retina, which is light-sensitive. Furthermore, this suppressive effect of light was effective only at light intensities down to 1 lux, and not at 0.4lux. This step-transition is a characteristic of the retinal dark-light switch, suggesting a role for this switch in controlling eye growth.