Hayasaka N et al. (2010), Differential contribution of rod and cone circa...

XB-ART-42537

PLoS One 2010 Dec 17;512:e15599. doi: 10.1371/journal.pone.0015599.

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Differential contribution of rod and cone circadian clocks in driving retinal melatonin rhythms in Xenopus.

Hayasaka N , LaRue SI , Green CB .

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Although an endogenous circadian clock located in the retinal photoreceptor layer governs various physiological events including melatonin rhythms in Xenopus laevis, it remains unknown which of the photoreceptors, rod and/or cone, is responsible for the circadian regulation of melatonin release. We selectively disrupted circadian clock function in either the rod or cone photoreceptor cells by generating transgenic Xenopus tadpoles expressing a dominant-negative CLOCK (XCLΔQ) under the control of a rod or cone-specific promoter. Eyecup culture and continuous melatonin measurement revealed that circadian rhythms of melatonin release were abolished in a majority of the rod-specific XCLΔQ transgenic tadpoles, although the percentage of arrhythmia was lower than that of transgenic tadpole eyes expressing XCLΔQ in both rods and cones. In contrast, whereas a higher percentage of arrhythmia was observed in the eyes of the cone-specific XCLΔQ transgenic tadpoles compare to wild-type counterparts, the rate was significantly lower than in rod-specific transgenics. The levels of the transgene expression were comparable between these two different types of transgenics. In addition, the average overall melatonin levels were not changed in the arrhythmic eyes, suggesting that CLOCK does not affect absolute levels of melatonin, only its temporal expression pattern. These results suggest that although the Xenopus retina is made up of approximately equal numbers of rods and cones, the circadian clocks in the rod cells play a dominant role in driving circadian melatonin rhythmicity in the Xenopus retina, although some contribution of the clock in cone cells cannot be excluded.

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Species referenced: Xenopus laevis
Genes referenced: clock

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	Figure 1. XCLΔQ-GFP expression in the specific cell-types in the transgenic photoreceptors.A. An image of a section from the XOP-XCLΔQ-GFP tadpole retina. XCLΔQ-GFP is observed only in the cell bodies and inner segments of the rod photoreceptor cells. B. In a CAR-XCLΔQ-GFP transgenic retina, GFP accumulates only in the cone photoreceptor cells. CB: photoreceptor cell body; OS: photoreceptor outer segment; IR: inner retina; RPE: retinal pigment epithelium. The scale bar indicates 10 µm.
	Figure 2. Melatonin release from the XOP-XCLΔQ-GFP and the CAR-XCLΔQ-GFP transgenic eyecups and wild-type controls.Each pair of eyecups was prepared from individual tadpoles and flow-through culture was performed for 5 days. Media fractions were collected every four hours, and assayed for melatonin by RIA. Each line represents melatonin release from a pair of eyecups. A. Melatonin release rhythms in the individual XOP-XCLΔQ-GFP eyecups (n = 5) and wild-type controls (n = 7). As compared to the wild-type eyes that demonstrate melatonin release in a circadian manner for five days, the majority of the transgenic eyes do not show significant circadian melatonin rhythmicity. B. Melatonin rhythms in the CAR-XCLΔQ-GFP (n = 6) and wild-type eyecups (n = 6). With some exceptions, eyecups release melatonin in a circadian manner.
	Figure 3. Total melatonin levels of the transgenic eyes and wild-type controls were comparable.Average of all fractions from the transgenic and wild-type eyes was calculated for the two different genotypes (XOP and CAR). Values on the figure are average melatonin content (picograms per 4hr) +/− SEM. A. The XOP transgenic (n = 25) vs. wild-type eyes (n = 37). B. The CAR transgenic (n = 17) vs. wild-type eyes (n = 73).
	Figure 4. Expression levels of the two different transgenes are comparable.After flow-through culture was complete, each pair of eyes was collected, RNA was extracted and real-time PCR was performed on GFP to compare relative levels of transgene expression. The average GFP levels from the XOP (n = 25) and CAR (n = 26) transgenic eyes were comparable and the difference was not statistically significant. Values are average relative GFP expression levels +/− SEM.
	Figure 5. Arrhythmic melatonin secretion correlates with mRNA levels of XOP-XCLΔQ, but not CAR-XCLΔQ.qPCR was performed on GFP as described in Figure 4. The GFP mRNA levels from the arrhythmic and rhythmic animal groups in each of the two transgenic animals were averaged. A. Comparison of GFP levels between rhythmic (XOP-R; n = 13) and arrhythmic (XOP-AR; n = 12) groups in the XOP transgenics (P<0.05, Student t-test). B. Rhythmic (CAR-R; n = 8) and arrhythmic (CAR-AR; n = 11) groups in CAR transgenic eyecups expressed comparable levels of GFP. Values are average relative GFP expression levels +/− SEM.

References [+] :

Alvarez-López, Altered endogenous activation of CREB in the suprachiasmatic nucleus of mice with retinal degeneration. 2004, Pubmed