Ž . Developmental Brain Research 117 1999 109–116 www.elsevier.comrlocaterbres Research report Ontogeny of circadian and light regulation of melatonin release in Xenopus laeÕis embryos Carla B. Green a, ) , Mei-Ying Liang b , Brooke M. Steenhard b , Joseph C. Besharse b a Department of Biology, NSF Center for Biological Timing, UniÕersity of Virginia, CharlottesÕille, VA 22903, USA b Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA Accepted 27 July 1999 Abstract The retinal photoreceptors of Xenopus laeÕis contain a circadian clock that controls the synthesis and release of melatonin, resulting in high levels during the night and low levels during the day. Light is also an important regulator of melatonin synthesis and acts directly to acutely suppress melatonin synthesis during the day and indirectly to entrain the circadian clock. We examined the development of circadian and light regulation of melatonin release in Xenopus retinas and pineal glands. Pineal glands are capable of making measurable melatonin in culture soon after they evaginate from the diencephalon at stage 26. In cyclic light, the melatonin rhythms are robust, with higher overall levels and greater amplitudes than in constant darkness. However, the rhythm of melatonin release damps strongly and Ž . quickly toward baseline in constant darkness. Similar results are observed in older stage 47 embryos, indicating that cyclic light has a positive effect on melatonin synthesis in this tissue. Optic vesicles dissected at stage 26 do not release melatonin in culture until the second or third day. It is weakly rhythmic in cyclic light, but in constant dark it is released at constitutively high levels throughout the day. By stage 41, the eyes release melatonin rhythmically in both cyclic light and constant darkness with similar amplitude. Our results show that Xenopus embryos develop a functional, photoresponsive circadian clock in the eye within the first few days of life and that rhythmic melatonin release from the pineal gland at comparable stages is highly dependent on a light–dark cycle. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Retina; Pineal gland; Development; Photoreceptor 1. Introduction Xenopus laeÕis retinas synthesize and release melatonin rhythmically with high levels at night and low levels wx during the day 4 . Melatonin is synthesized by cells within w x the photoreceptor layer 12 and is regulated both by light w x wx 11,28 and by a circadian clock 4 , also located within the w x photoreceptor layer 13 . Light acts both to acutely sup- press melatonin during the day and cycles of light also w x entrain the circadian clock 4,5,11 . Melatonin synthesized Ž in the retina acts as a local neuromodulator reviewed in w x. 14 , is rapidly broken down in the retina, and probably does not contribute significantly to circulating melatonin wx levels 9 . In most vertebrates, the primary source of w x circulating melatonin is the pineal gland 38 . In Xenopus and other non-mammalian vertebrates, the pineal gland contains cells that resemble retinal photoreceptor cells and ) Corresponding author. Fax: q1-804-982-5626; e-mail: cbg8b@virginia.edu w x is directly photosensitive 16,20,32,50 . In addition, pineal glands of some, but not all, non-mammalian vertebrates have been shown to contain endogenous circadian clocks that can control rhythmic melatonin release in vitro w x 6,8,19,21,26,30,48,52 . Melatonin and enzymes involved w x in its biosynthesis are present in Xenopus embryos 1,2,49 and melatonin of pineal gland origin is involved in the w x blanching of skin color 3,15 . Very little is known about control of melatonin synthesis or circadian clock function in the pineal gland of Xenopus. Xenopus has been used extensively for embryological studies due to its rapid development and its ability to survive transplant surgeries and explant culture experi- ments. The morphological details of development have w x been well described 36 , but the ontogeny of the circadian system has not been characterized. The pineal anlage evaginates from the diencephalon beginning at stage 26, which occurs on approximately the second day of develop- ment. It continues to grow, flatten, and differentiate over the next several days. The eyes develop first as optic 0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0165-3806 99 00109-1