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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 6, 2004 as doi:10.1096/fj.03-0544fje. |
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* Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA;
Department of Molecular Medicine, Beth Israel Deaconess Center, Harvard Medical School, Boston, Massachusetts, USA;
Centre for Reproduction, Endocrinology and Diabetes, School of Biomedical Sciences, Kings College London, UK; and
Department of Anatomy and Neurobiology, Boston University Medical School, Boston, Massachusetts, USA
2 Correspondence: Nadia Danilova, Department of Biology, 68-265, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA. E-mail: ndanilov{at}mit.edu
SPECIFIC AIMS
Melatonin, a hormone produced at night, synchronizes other circadian rhythms by acting on specific G-protein coupled receptors. Melatonin receptors are more highly expressed in developing embryos and neonates than adults, though the role of melatonin in development is not known. The aim of this study was to elucidate the role of melatonin in development.
PRINCIPAL FINDINGS
1. Expression of melatonin receptors, MT2 in particular, is upregulated in zebrafish embryo
We have found, that in the brain of adult zebrafish, as in other species, the MT1 subtype is more highly expressed than MT2. However, in the embryo, the MT1/ MT2 ratio is different from the adult fish in that both subtypes show equally high expression. Receptor mRNA can be first detected at 
18 h post fertilization (hpf) and quickly becomes widespread suggesting that many embryonic tissues can be a target for melatonin. By 24 hpf, very strong expression is seen in the brain, and weaker staining in the dorsal aorta, while in the tail and the trunk expression decreases. It is further reduced by 36 h and becomes mostly restricted to the paraventricular regions of the brain.
The appearance of melatonin receptor mRNA coincides with the onset of melatonin synthesis.
2. Melatonin stimulates cell proliferation in the embryo
We used a nucleotide analog 5-bromo-2-deoxy-uridine (BrdU) to label proliferating cells in zebrafish embryos and evaluated the rate of proliferation by immunoassay. We observed that the rate of proliferation was not influenced by melatonin before the beginning of melatonin receptor expression, however it increased significantly in embryos treated with melatonin at the time they start to express melatonin receptors (18.5 hpf, Fig. 1
a). Melatonin effect decreased in older embryos, with reduction of melatonin receptor expression (38 hpf, Fig. 1b
). These data suggest that one of the functions of melatonin is to regulate the rate of cell proliferation in dependence with photoperiod.
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3. Embryos treated with melatonin develop faster
Starting from 18 hpf, when melatonin receptor expression begins, the developmental pace of melatonin-treated embryos increases as measured by several morphological and functional criteria (Fig. 2
a–e). This effect is dose-dependent as indicated by two staging methods, the frequency of muscle contraction (data not shown) and hatching (Fig. 2e
). The effect is evident at 10–9 M and has plateau from 10–8M to 10–4 M, as would be expected from the saturation of high affinity melatonin receptors. To compare the sensitivity to melatonin at different developmental stages, we pulse-treated embryos with the hormone (10–8M, for 1.5 h). Acceleration of development was evident from 18.5 hpf, and it decreased after 36 hpf (Fig. 2f
). Our data show that both the increase in proliferation rate and the acceleration of development in response to melatonin occur at the same time-window, suggesting a possible causal link between the two effects.
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4. Melatonin receptors mediate the effect of melatonin
To confirm that melatonin accelerates development by acting through specific receptors, we studied the effects of melatonin receptor agonists and antagonists. Of the two melatonin receptor agonists, (–) and (+) AMMTC, the (–) enantiomer accelerated development as well as melatonin, and much more effectively than the (+) enantiomer. AMMTC is a non-subtype selective melatonin receptor agonist; the (–) enantiomer mimics the active conformation of melatonin and has a receptor affinity similar to melatonin while the (+) enantiomer has >100-fold lower affinity. Luzindole, a melatonin receptor antagonist, blocked the effect of melatonin on zebrafish development. It is noteworthy that when applied alone, luzindole had a small inhibitory effect on zebrafish development, presumably antagonizing endogenous melatonin.
5. The effect of MT2-specific ligands on zebrafish development
Subtype selective melatonin receptor ligands are necessary to elucidate the role of the receptor subtypes in the melatonin effect on development. At present, there are compounds with some MT2 receptor selectivity, but not MT1 selective ligands. IIK7, an agonist with 90 times higher affinity for the MT2 receptor subtype than for MT1, accelerated zebrafish development and its effect was comparable to that of melatonin. K185, an antagonist having 140 times higher affinity for the MT2 subtype than for MT1, inhibited melatonin’s effect on development. The effects of MT2 specific ligands suggest a role for this subtype in the melatonin effect on development. Our data on 4-P-PDOT also support this notion. Both radioligand binding and functional measures of receptor activation indicate that 4-P-PDOT is a selective MT2 receptor ligand. 4-P-PDOT was initially reported to act as a melatonin receptor antagonist in the retina, but has been shown to act as a partial agonist on cells expressing a high density of MT2 receptors. It was reported to have no or only a low-affinity antagonist action on MT1 expressing cells. Therefore, one might expect 4-P-PDOT to act as an agonist on tissues with high expression of MT2, which was the case for zebrafish embryos from 18 to 36 hpf. A low concentration of 4-P-PDOT (10–8M), unlikely to activate MT1 receptors, accelerated zebrafish development to the same extent as melatonin. Taken together, our data on melatonin receptor ligands point to the possibility that MT2 plays a major role in mediating melatonin acceleration of development. Further experiments are needed to conclusively establish the melatonin receptor subtype responsible for accelerating zebrafish embryo development.
CONCLUSIONS
The main finding of our work is that melatonin stimulates cell proliferation in the embryo and accelerates zebrafish development acting through specific melatonin receptors. The onset of these effects coincides in time with the beginning of endogenous melatonin production and with the upsurge of melatonin receptor expression.
This effect suggests that a new and previously unrecognized role of melatonin may be to stimulate cell proliferation in developing tissue at nighttime, when there is no danger of cell damage by ultraviolet (UV) light.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0544fje; doi: 10.1096/fj.03-0544fje 
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