Measurement of orcadian rhythms in pineal gland and retina of embryonic xenopus laevis

C. B. Green, J. C. Bcsharscl

Research output: Contribution to journalArticle

Abstract

Purpose. We have previously shown that a circadian clock is present in the retinal photoreceptor cells of post-metamorphic Xenopus laevis. This clock regulates many aspects of retinal physiology including synthesis and release of the ncuromodulator melatonin and expression of the genes encoding tryptophan hydroxylase (TPH) and nocturnin. The molecular mechanism of clock function is not known. In order to address this question more fully, we have initiated studies of circadian rhythmicity in embryonic Xenopus. Use of embryonic tissue is potentially advantageous because it is generally more amenable to experimental manipulation. Methods. Xenopus embryos were raised in 12 hr light/12 hr dark cyclic light. At the appropriate stage, pineal glands or eyes were dissected from the embryos and transferred to culture medium. The eyes were further dissected by removing the anterior portion, including the lens, thereby producing a miniature "eyecup" with the retina exposed to the culture medium. Tissue was cultured at 2I°C in cyclic light or constant darkness for several days and medium collected at intervals for assay of melatonin. Whole mount in situ hybridizations were performed using digoxygenin labelled riboprobes. Results. Isolated pineal glands synthesize melatonin rhythmically in culture in cyclic light as early as siage 2ft and in consiant darkness by stage 32. Consistent with this finding, we observed the inRNA for TPH, one of the melatonin synthetic enzymes, in the pineal at these stages. Low levels of melatonin can also be measured from the eyecups dissected at stage 35. Conclusions. This data demonstrates that the Xenopus pineal, which is a photoreceptive organ, contains an endogenous circadian clock that is detectable early in development. Both the pineal and the eye can be isolated from embryos and maintained in vitro. This provides an important new system in which to study the circadian clock and its rhythmic outputs.

Original languageEnglish (US)
Pages (from-to)S53
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number4
StatePublished - Dec 1 1997

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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