Norio Ishida (Prime Senior Researcher) and Toshifumi Minamoto (former Post-Doctoral Research Scientist at Ishida Group of Clock Gene and currently a Senior Project Researcher at Research Institute for Humanity and Nature), the Institute for Biological Resources and Functions (Director: Masanao Oda) of the National Institute of Advanced Industrial Science and Technology (AIST) (President: Tamotsu Nomakuchi), jointly obtained the physiological- and genetic-level findings for a biological clock in C. intestinalis.  This study was performed in cooperation with Kyoto University, Hokkaido University, Tohoku University, and Tokyo Medical and Dental University.

Ascidians or sea squirts, which are members of invertebrate chordates that are closer to humans than Drosophila, have been frequently studied as animal models in developmental biology.  However, the existence of a biological clock in ascidians has not been reported to date.  In our experiment, ascidians were maintained in a tank under light-dark or constant-dark conditions, and the oxygen consumption was measured.  We observed a rhythmic pattern in the oxygen consumption in both light-dark and constant-dark conditions (Fig. 1).  In the analysis of the expression levels of all the genes (about 22,000), 388 genes showed a 24-hour biological rhythm.  After reversing the light-dark cycles, 4 of these genes were selected for detailed analysis of gene-expression levels.  The cycles of 3 genes were reset in response to changes in the new light-dark cycle, and these genes showed a new cyclic expression pattern.  This was the first genetic-level evidence on the existence of a biological clock in ascidians.

This result was published in the October 26 issue of "The Journal of Biochemistry," an international journal of biochemistry.

Fig. 1  Rhythmic pattern in the oxygen consumption of ascidians

Photograph of C. intestinalis (length, 13 cm)

With the aging population and increasingly hectic 24-hour lifestyle in advanced countries, the number of patients with sleep disorders is increasing.  Further, circadian-rhythm sleep disorders and winter depression, which are caused by excessively controlled organizational systems and rapid computerization, have now been recognized as social problems.  However, there is very little information about the molecular mechanism of the human biological clock, which is the underlying factor in these disorders.

In Drosophila and human, identified clock genes include Clock, Bmal, and period, but there is little information on the precise mechanisms explained for the biological clock in humans.  Ascidians, which are members of non-vertebrate chordates that are closer to humans than Drosophila, have been frequently used as animal models in developmental biology.  However, the existence of a biological clock in ascidians has not been reported so far.  The elucidation of a biological clock in ascidians will be important for understanding the mechanism of the human biological clock.

Many living organisms use their biological clocks to adapt themselves to various cyclical phenomena in the environment.  To elucidate the molecular mechanisms of biological clocks, AIST has studied mammals, Drosophila, as well as ascidians by using molecular biological techniques.

C. intestinalis, which is one of the earilest evolved forms of chordates, has been used as a model organism in developmental biology.  A number of findings have been deduced from the studies on this organism, and abundant genomic and expressed sequence tag heredity data for C. intestinalis have been obtained.  However, the biological clock of C. intestinalis has been rarely studied, and the existence of the clock itself has not been confirmed.

Because ascidians are positioned midway between mammals and invertebrates, we started studying the molecular mechanism of the ascidian biological clock, which may be useful in understanding the evolution and the mechanism of the human biological clock.

We examined the recently published draft DNA sequence of C. intestinalis, but we could not identify any DNA sequences for the known clock genes such as Clock, Bmal and period that could have been evolutionarily preserved from invertebrates to mammals.

Then, we grew C. intestinalis in a laboratory tank and measured the oxygen consumption in the light-dark condition and the constant-dark condition.  We consequently detected a rhythm that peaked at the early hours of night (Fig. 1).  This rhythm was maintained after shifting to constant-dark condition.

The ascidians were maintained in the tank under 12-hour light/dark cycles, and their gene-expression levels (the amount of mRNA formed) were determined.  Microarray chips developed by the Hokkaido University group were used for comprehensive genetic analysis of approximately 22,000 genes.  The result revealed that 388 genes showed a 24-hour biological rhythm.

Next, we conducted the following experiment to examine the detailed mechanism of the rhythm that was maintained even under the constant-dark condition (circadian rhythm).  The ascidians were entrained over 3 cycles of 12-hour light-dark phases, which were followed by the constant-dark condition.  The measurement of the gene-expression levels in the cells (8 times at 3-hour intervals) was started on the second day after shifting to the constant-dark condition (this experimental condition was denoted by the ▼ symbol in Fig. 2).  To change the light-dark cycle condition, the second half of the third cycle was reversed from dark to light phase and the gene-expression levels were measured (this experimental condition was denoted by the △ symbol in Fig. 2).

Fig. 2  Conditions in which the gene-expression levels were measured Regular condition: Measurement of gene-expression levels was started on the second day after completion of light-dark cycles for three days (▼). Reversed light-dark condition: The dark phase of the third light-dark cycle was reversed to a light phase, and gene-expression levels were measured on the second day after the exposure to the extended light cycle (△).

Fig. 3 shows the results of the experiment.  As shown by the ■ symbols, even after induction of the constant-dark environment after entrainment in light-dark cycles, the gene expression continued to change periodically, and the circadian rhythm was confirmed.  As shown by the □ symbols in Fig. 3, the rhythmic pattern of the expressions of genes A, B, and C changed in response to the new light-dark cycles.  Genes A, B, and C seemed to be reset by light and respond to the new cycles.

Fig. 3  Measurement results for gene-expression levels ■ The oscillation in gene-expression levels continued even in the constant-dark condition after entrainment in light-dark cycles. □ The gene-expression patterns changed in response to the new cycles induced by reversing the light-dark cycle (Genes A, B, and C).

As the biological clock is reset by light, this is the first report on the gene-level evidence for the existence of a biological clock in ascidians.  The analysis of the draft DNA sequence of ascidians did not reveal any DNA sequences for the known clock genes; therefore, the clock system in ascidians might be completely different from those in Drosophila and mammals.

To further study the molecular mechanism of the ascidian biological clock, we will explore the functions of the genes that showed changes in the periodic gene-expression pattern in response to environmental changes.  Further, if these genes alone cannot explain the mechanism, we will continue studies to identify the principal gene in the biological clock of ascidians.