LONDON - Fish embryos inherit a circadian clock from their mothers, and are able to respond to light cues from day one of embryogenesis, new research has shown.

The discovery disproves one of the disputed dogmas of circadian rhythm research: that a brain is required in order to have a circadian clock. The study also demonstrates, astonishingly, that fish embryos only two hours old have some way of detecting light and dark.

Vincent Laudet, professor of biology at the Ecole Normale Superieure at CNRS in Lyon, France, told BioWorld International: "These findings could lead to the development of drugs which could play a role in circadian rhythm problems, including jet lag, sleep disorders, depression, aging and problems associated with shift working." He and his group are looking for partners to help them develop their work further.

A description of the experiments conducted by Laudet and his colleagues in Lyon, in collaboration with Christine Thisse and Bernard Thisse of the Institut de Genetique et de Biologie Moleculaire et Cellulaire in Strasbourg, France, appears in the July 14, 2000, issue of Science. The title of the paper is "An Inherited Functional Circadian Clock in Zebrafish Embryos."

Ten years ago, Laudet's group and others discovered the gene known as Rev-erba, which encodes a nuclear hormone receptor. Since then, they have been trying to identify the ligand for this receptor and establish its function.

By chance, they found that Rev-erba is expressed in a circadian fashion in adult zebrafish, and further studies showed that the same was true in zebrafish embryos. Laudet said, "This was a big surprise because it has been assumed in the past that a brain is necessary in order to have a circadian rhythm, and so you would not expect to find such a rhythm in an early embryo. This finding induced us to test the hypothesis that the clock existed very early in embryogenesis."

The group went on to identify a gene in zebrafish that was homologous to genes in Drosophila and the mouse that are known to be involved in controlling the circadian clocks of these species. The gene is called PER3 and its protein product per3.

The zebrafish embryo is relatively large and completely transparent. When the researchers used a probe for messenger RNA derived from PER3, coupled to a chemical that induces a colored reaction, they were able to see very clearly and easily where expression was taking place, and to what extent. Working with zebrafish embryos raised under a cycle of 14 hours of light and 10 hours of darkness, they found that expression of PER3 followed an apparent circadian rhythm, coming on at the end of the dark period and going off at the end of the light period.

For this pattern to fulfill the criteria of a true circadian rhythm, Laudet explained, it needed to continue even in constant light or constant darkness. Secondly, a true circadian rhythm can be synchronized with light.

Further experiments proved this was the case. The rhythmic expression of PER3 continued even in continuous light or continuous darkness. "In addition," Laudet said, "when we shifted the light/dark pattern by eight hours - just as if we had sent the fish to New York - the embryos reset their clock, much more happily than humans do. This means that the embryos must be able to 'see' the light, which was a real surprise."

The team showed that the embryos could respond to light in this way as early as the first day of development.

To their fascination, they also discovered that the embryos inherited their clock from their mothers. In this species, transcription of embryonic genes does not begin until the 10th cell cycle. Yet Laudet and his colleagues found that PER3 was present in two-cell embryos, and was therefore of maternal origin.

When they compared embryos fertilized at 8 a.m. with those fertilized at midnight, they found that although their developmental stages were different - as would be expected - their circadian clocks were exactly the same.

Laudet explained, "The embryo 'knows' from the amount of Per3 that it is loaded with at the beginning of embryogenesis if it is the day or if it is the night. This means that the clock is inherited by the embryo from the mother." Further experiments, using polymerase chain reaction, confirmed that Per3 levels in oocytes in adult zebrafish were at a maximum at 8 a.m., and at a minimum at midnight.

The data presented in Science demonstrate that a classical circadian clock can exist in an embryo that has no brain. "This confirms other studies which have shown that isolated cells in culture can have a circadian clock, but the problem with observations such as those is that a culture of cells is an artificial system," Laudet said.

One question everyone working on the project would like to see answered is how an embryo that has neither brain nor eyes can 'see' the light. Laudet speculated, "We know that in vertebrates there are molecules called cryptochromes which are important in circadian rhythms. The genes encoding these molecules are closely related to plant genes encoding enzymes known as photolyases. The enzymatic activity of these proteins is photo-dependent, so we suspect that cryptochromes play a role, but as yet we have no proof."

Next, Laudet and his colleagues plan to examine the inheritance of other gene products involved in regulation of circadian clocks. The group also wants to find out what the relationship is between developmental timing and the circadian clock. "We want to know whether the circadian clock needs proper development, and whether proper development needs a circadian clock. If you put embryos in constant dark or constant light, will their development be affected?" Laudet added.

The group also is continuing to search for a ligand to Rev-erba by screening molecules that are known to play a role in circadian rhythms. Laudet concluded, "The fact that we have found a nuclear hormone receptor implicated in circadian rhythms may lead to new strategies for the development of drugs that may be helpful in the treatment of jet lag, sleep disorders, depression, aging and problems associated with shift working, for example."

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