- Email: [email protected]
Setting the clock in Madrid
Piecing together the clock The above questions, and others, constituted the centre of debate during the recent ‘Molecular Clocks’ meeting organized by the Juan March Foundation*. M. Menaker (Charlottesville, USA) opened the
Setting the clock in Madrid
MEETING REPORT
Circadian and seasonal rhythms are central to most biological systems, from daily oscillations in plant photosynthesis to hormone secretion and annual breeding cycles in mammals. All animals have an endogenous clock or pacemaker that, independently from the day–night cycle, generates circadian rhythms in physiology and behaviour. The clock is predicted to consist of three functional components: • an input pathway for entrainment to light–dark cycles; • an autonomous circadian pacemaker that generates the oscillation; • an output pathway that directs the expression of physiological rhythms. For years, researchers thought that these regulatory systems required an intact tissue organization and relied upon intercellular communications. Today, we know that each cell comprising the pacemaker has endogenous oscillatory properties and contains an autonomous clock. Remarkable strides have characterized the circadian clock field during the past 10 years. Much attention has been given to the search for genes responsible for the clock function. Clock genes have been cloned in Drosophila, Neurospora, zebrafish and mammals. An emerging common feature is that most clock genes encode proteins with the structural characteristics of transcription factors, although there are notable exceptions. It is evident that clock molecules operate within regulatory networks where autoregulatory feedback loops play a central role. The example of Drosophila PER and TIM – factors encoded by the period and timeless genes, respectively – represents a paradigm in the field. The identification of molecular clock components has provided powerful tools to address fundamental biological questions such as: • which cells contain clocks? • when and how does the clock start ticking? • how is it able to anticipate the light–dark cycle, and • how is light able to directly influence clock function?
Nicholas S. Foulkes, José R. Naranjo and Paolo Sassone-Corsi meeting with an overview of the effect of light on circadian rhythms and seasonal reproductive capacity. Extraretinal photoreceptors, which are present in all other vertebrates, have not been found in mammals, so all responses to light are thought to be mediated by the retina. However, recent results obtained using albino hamsters indicate that circadian, retinal photoreceptors, by themselves, are not sufficient to support the reproductive response to light. These results predict the existence of an asyet-uncharacterized class of photoreceptor that would mediate reproductive responses to photoperiods. Evidence for a new type of photoreceptor comes from the work of R. Foster (London, UK). This circadian photoreceptor is likely to differ from photoreceptors of the visual system as the circadian response to light is conserved in transgenic mice that have degeneration of rod and cone photoreceptors. Important additional insights on how light might directly affect the circadian clock is given by experiments described by J. Hoeijmakers (Rotterdam, The Netherlands). Two molecules have been identified in the mouse, CRY1 and CRY2, that are homologues of plant blue-light receptors (cryptochromes) and photolyases. One intriguing feature of mammalian CRY proteins is their presence in tissues that are not commonly thought to be light sensitive. Strikingly, targeted disruption of the genes encoding these two molecules results in aberrant circadian locomotor activity in the mouse. Furthermore, CRY1–CRY2 double mutants are completely arhythmic, indicating that CRY proteins are essential components of the endogenous clock. A CRY homologue has also been identified in Drosophila. M. Rosbash (Waltham, USA) reported on its cyclic expression and tissue distribution. Transgenesis and studies in cultured cells confirm the central role played by CRY in Drosophila photoreception. In a complementary study, R. Stanewsky (Resensburg, Germany) performed an extensive mutagenesis in the fly and identified a CRY mutant that displays lack of oscillation in the period and timeless genes.
trends in CELL BIOLOGY (Vol. 9) September 1999
The pineal gland is the anatomical structure that directs the circadian synthesis of the hormone melatonin (Fig. 1). It is light sensitive in lower vertebrates and is thought to have lost this property during mammalian evolution, although neonatal rat pineal cells in culture appear to be light responsive (M. Menaker). S. H. Snyder (Baltimore, USA) described a day–night subtractive hybridization approach that they used to identify genes expressed differentially day and night in the rat pineal. One interesting find is the dramatic oscillation in the expression of Patched 1 (PTC-1), a tumour suppressor homologue of the Drosophila segment-polarity gene patched that acts as a receptor for hedgehog during embryonic development. Another protein that oscillates in the pineal gland is DREAM, a transcription factor described by J. R. Naranjo (Madrid, Spain) that is under the control of Ca2+ signalling.
*Molecular Clocks; Madrid, Spain; 10–12 May 1999. Organized by José R. Naranjo and Paolo Sassone-Corsi.
Transcription factors The search for genes involved in clock function in various organisms has led to the identification of a number of new transcription factors. In the Neurospora circadian clock, where the frequency gene plays a central role, additional elements have been found by J. C. Dunlap (Hanover, USA). One of them, WC-1 (white collar 1) displays intriguing sequence similarity with the mammalian BMAL proteins. BMALs are partners of CLOCK, the only bona fide clock protein in mammals to date’ identified by J. Takahashi (Evanston, USA) by mutational screening in the mouse. The common feature of these transcription factors is the presence of PAS domains, a structural motif involved in protein– protein interactions and so named from the three initial members of the PAS family: PER, Arnt (a dimerization partner of the dioxin receptor) and Singleminded. The search for mammalian homologues of the Drosophila clock genes has led to the identification of three per genes (mper1, mper2 and mper3) and one tim gene in the mouse, suggestive of greater complexity in the regulation and combinatorial
Nicholas Foulkes and Paolo Sassone-Corsi are in the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRSINSERM-Université Louis Pasteur, B. P. 163, 67404 Illkirch, Strasbourg, France; and José Naranjo is at the Cajal Institute, Madrid, Spain. E-mail: paolosc@igbmc. u-strasbg.fr
0962-8924/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved.
371
PII: S0962-8924(99)01622-0
meeting report
FIGURE 1 The pineal gland is one of the most remarkable neuroendocrine organs in the body. It is a small gland located in the centre of the skull between the two cerebral hemispheres. Four centuries ago, Descartes suggested that it was the seat of the soul since it represents the only nonpaired structure in the human brain. In lower vertebrates, the pineal gland responds to light (one of its popular names is ‘the third eye’) and possesses an endogenous clock function. In mammals, central clock functions classically are thought to be located in a separate hypothalamic structure, the suprachiasmatic nucleus (SCN). Light stimuli reach the SCN indirectly via the retinohypothalamic pathway. Through a multisynaptic pathway, neurones of the SCN project to the intermediolateral cell column of the spinal cord, which contains cell bodies that innervate the superior cervical ganglion. Sympathetic postganglionic neurons then ascend to innervate the pineal gland. This is a drawing by Santiago Ramon y Cajal (1852–1934) that shows with remarkable precision and clarity these innervations. The drawing is part of a collection kept at the Cajal Institute in Madrid, Spain (Legado Cajal, Consejo Superior de Investigaciones Cientificas).
functions of these factors in mammals. The results presented by M. Antoch (Evanston, USA) using the clock mutant mice demonstrate that CLOCK directly regulates the expression of per genes in vivo. This is consistent with experiments by C. Weitz (Boston, USA) who transfected the mper1 gene promoter in cultured cells and studied its regulation by CLOCK and BMAL proteins. An important feature of clock genes in mammals is their generalized expression in various tissues. The tissuespecific regulation of the three per genes was studied by S. Reppert (Boston, USA), who described also the intracellular localization of the protein products. Importantly, he noted the presence of a dimerization code between the three PER proteins and TIM, where not all combinations are possible, suggestive of additional levels of regulation. M. Hastings (Cambridge, UK) described the patterns of PER and TIM protein expression during the circadian cycle in the suprachiasmatic nucleus (SCN), the anatomical centre of the endogenous clock. That other elements are still missing from the clock puzzle is demonstrated by the results presented by M. Young (New York, USA) in Drosophila. Using a differential display approach, Young and colleagues have isolated vrille (vri), a transcription factor previously identified as having a role in development. The VRI protein shows homology to DBP, a transcription factor that oscillates in the liver and in the SCN. The expression of vri is synchronous with that of per and tim and is low in flies mutated for Clock, suggesting that vri is regulated by the transcriptional regulatory loops formed by the other clock proteins. A pace-setting trend? A major issue of clock research has been the identification of anatomical structures containing independent
pacemakers. Results in zebrafish presented by N. S. Foulkes and P. SassoneCorsi (Strasbourg, France) indicate that expression of the Clock gene homologue oscillates not only in the two defined clock structures – the eye and pineal gland – but also in peripheral tissues such as the heart and kidney. A similar pattern was also observed for the two zebrafish CLOCK partners, BMAL1 and BMAL2. The finding is even more striking as the oscillation is maintained in organ ex vivo cultures of these tissues. The presence of peripheral oscillators in Drosophila was demonstrated elegantly by P. Hardin (Houston, Texas) in the fly antenna by looking at the circadian response of electrical activity induced by odorant stimuli. If oscillators are present in peripheral tissues, then there is a chance that they might also be revealed in single cells. Experiments conducted by U. Schibler (Geneva, Switzerland) indicate the presence of circadian oscillations in gene expression in cultured immortalized fibroblasts in response to serum. This fibroblastic clock is independent of the cell cycle but is influenced in different ways by agents acting on various intracellular signalling pathways. Time to take stock Our understanding of the functioning of the circadian clock is progressing by leaps and bounds, and the future will undoubtedly hold many surprises. Crucial questions are: • what is the biological role of peripheral clocks? • how does light entrain the clock? • how do the molecular components of the clock work together, and • how are they modulated by intracellular pathways? Hopefully, we will have the answers in a forthcoming meeting.
http://tto.trends.com New articles published recently in Technical Tips Online include: A trilogy of Core Protocols: • Parchaliuk, D. L., Kirkpatrick, R. D., Simon, S. L., Agatep, R. and Gietz, R. D. (1999) Yeast two-hybrid system: part A –screen preparation (p01616); part B – screening procedure (p01713); part C – characterizing positives (p01714) (http://tto.trends.com) and two new Technical Tips: • Kuschak, T. I., Paul, J. T., Wright, J. A., Mushinski, J. F. and Mai, S. (1999) FISH on purified extrachromosomal DNA molecules (http://tto.trends.com) t01669 • Coleman, A. E., Angkustsiri, K. and Janz, S. (1999) Use of B1-repeat-supplemented Cot-1 DNA to enhance background suppression in FISH (http://tto.trends.com) t01769
372
trends in CELL BIOLOGY (Vol. 9) September 1999
Setting the clock in Madrid
MEETING REPORT
Circadian and seasonal rhythms are central to most biological systems, from daily oscillations in plant photosynthesis to hormone secretion and annual breeding cycles in mammals. All animals have an endogenous clock or pacemaker that, independently from the day–night cycle, generates circadian rhythms in physiology and behaviour. The clock is predicted to consist of three functional components: • an input pathway for entrainment to light–dark cycles; • an autonomous circadian pacemaker that generates the oscillation; • an output pathway that directs the expression of physiological rhythms. For years, researchers thought that these regulatory systems required an intact tissue organization and relied upon intercellular communications. Today, we know that each cell comprising the pacemaker has endogenous oscillatory properties and contains an autonomous clock. Remarkable strides have characterized the circadian clock field during the past 10 years. Much attention has been given to the search for genes responsible for the clock function. Clock genes have been cloned in Drosophila, Neurospora, zebrafish and mammals. An emerging common feature is that most clock genes encode proteins with the structural characteristics of transcription factors, although there are notable exceptions. It is evident that clock molecules operate within regulatory networks where autoregulatory feedback loops play a central role. The example of Drosophila PER and TIM – factors encoded by the period and timeless genes, respectively – represents a paradigm in the field. The identification of molecular clock components has provided powerful tools to address fundamental biological questions such as: • which cells contain clocks? • when and how does the clock start ticking? • how is it able to anticipate the light–dark cycle, and • how is light able to directly influence clock function?
Nicholas S. Foulkes, José R. Naranjo and Paolo Sassone-Corsi meeting with an overview of the effect of light on circadian rhythms and seasonal reproductive capacity. Extraretinal photoreceptors, which are present in all other vertebrates, have not been found in mammals, so all responses to light are thought to be mediated by the retina. However, recent results obtained using albino hamsters indicate that circadian, retinal photoreceptors, by themselves, are not sufficient to support the reproductive response to light. These results predict the existence of an asyet-uncharacterized class of photoreceptor that would mediate reproductive responses to photoperiods. Evidence for a new type of photoreceptor comes from the work of R. Foster (London, UK). This circadian photoreceptor is likely to differ from photoreceptors of the visual system as the circadian response to light is conserved in transgenic mice that have degeneration of rod and cone photoreceptors. Important additional insights on how light might directly affect the circadian clock is given by experiments described by J. Hoeijmakers (Rotterdam, The Netherlands). Two molecules have been identified in the mouse, CRY1 and CRY2, that are homologues of plant blue-light receptors (cryptochromes) and photolyases. One intriguing feature of mammalian CRY proteins is their presence in tissues that are not commonly thought to be light sensitive. Strikingly, targeted disruption of the genes encoding these two molecules results in aberrant circadian locomotor activity in the mouse. Furthermore, CRY1–CRY2 double mutants are completely arhythmic, indicating that CRY proteins are essential components of the endogenous clock. A CRY homologue has also been identified in Drosophila. M. Rosbash (Waltham, USA) reported on its cyclic expression and tissue distribution. Transgenesis and studies in cultured cells confirm the central role played by CRY in Drosophila photoreception. In a complementary study, R. Stanewsky (Resensburg, Germany) performed an extensive mutagenesis in the fly and identified a CRY mutant that displays lack of oscillation in the period and timeless genes.
trends in CELL BIOLOGY (Vol. 9) September 1999
The pineal gland is the anatomical structure that directs the circadian synthesis of the hormone melatonin (Fig. 1). It is light sensitive in lower vertebrates and is thought to have lost this property during mammalian evolution, although neonatal rat pineal cells in culture appear to be light responsive (M. Menaker). S. H. Snyder (Baltimore, USA) described a day–night subtractive hybridization approach that they used to identify genes expressed differentially day and night in the rat pineal. One interesting find is the dramatic oscillation in the expression of Patched 1 (PTC-1), a tumour suppressor homologue of the Drosophila segment-polarity gene patched that acts as a receptor for hedgehog during embryonic development. Another protein that oscillates in the pineal gland is DREAM, a transcription factor described by J. R. Naranjo (Madrid, Spain) that is under the control of Ca2+ signalling.
*Molecular Clocks; Madrid, Spain; 10–12 May 1999. Organized by José R. Naranjo and Paolo Sassone-Corsi.
Transcription factors The search for genes involved in clock function in various organisms has led to the identification of a number of new transcription factors. In the Neurospora circadian clock, where the frequency gene plays a central role, additional elements have been found by J. C. Dunlap (Hanover, USA). One of them, WC-1 (white collar 1) displays intriguing sequence similarity with the mammalian BMAL proteins. BMALs are partners of CLOCK, the only bona fide clock protein in mammals to date’ identified by J. Takahashi (Evanston, USA) by mutational screening in the mouse. The common feature of these transcription factors is the presence of PAS domains, a structural motif involved in protein– protein interactions and so named from the three initial members of the PAS family: PER, Arnt (a dimerization partner of the dioxin receptor) and Singleminded. The search for mammalian homologues of the Drosophila clock genes has led to the identification of three per genes (mper1, mper2 and mper3) and one tim gene in the mouse, suggestive of greater complexity in the regulation and combinatorial
Nicholas Foulkes and Paolo Sassone-Corsi are in the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRSINSERM-Université Louis Pasteur, B. P. 163, 67404 Illkirch, Strasbourg, France; and José Naranjo is at the Cajal Institute, Madrid, Spain. E-mail: paolosc@igbmc. u-strasbg.fr
0962-8924/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved.
371
PII: S0962-8924(99)01622-0
meeting report
FIGURE 1 The pineal gland is one of the most remarkable neuroendocrine organs in the body. It is a small gland located in the centre of the skull between the two cerebral hemispheres. Four centuries ago, Descartes suggested that it was the seat of the soul since it represents the only nonpaired structure in the human brain. In lower vertebrates, the pineal gland responds to light (one of its popular names is ‘the third eye’) and possesses an endogenous clock function. In mammals, central clock functions classically are thought to be located in a separate hypothalamic structure, the suprachiasmatic nucleus (SCN). Light stimuli reach the SCN indirectly via the retinohypothalamic pathway. Through a multisynaptic pathway, neurones of the SCN project to the intermediolateral cell column of the spinal cord, which contains cell bodies that innervate the superior cervical ganglion. Sympathetic postganglionic neurons then ascend to innervate the pineal gland. This is a drawing by Santiago Ramon y Cajal (1852–1934) that shows with remarkable precision and clarity these innervations. The drawing is part of a collection kept at the Cajal Institute in Madrid, Spain (Legado Cajal, Consejo Superior de Investigaciones Cientificas).
functions of these factors in mammals. The results presented by M. Antoch (Evanston, USA) using the clock mutant mice demonstrate that CLOCK directly regulates the expression of per genes in vivo. This is consistent with experiments by C. Weitz (Boston, USA) who transfected the mper1 gene promoter in cultured cells and studied its regulation by CLOCK and BMAL proteins. An important feature of clock genes in mammals is their generalized expression in various tissues. The tissuespecific regulation of the three per genes was studied by S. Reppert (Boston, USA), who described also the intracellular localization of the protein products. Importantly, he noted the presence of a dimerization code between the three PER proteins and TIM, where not all combinations are possible, suggestive of additional levels of regulation. M. Hastings (Cambridge, UK) described the patterns of PER and TIM protein expression during the circadian cycle in the suprachiasmatic nucleus (SCN), the anatomical centre of the endogenous clock. That other elements are still missing from the clock puzzle is demonstrated by the results presented by M. Young (New York, USA) in Drosophila. Using a differential display approach, Young and colleagues have isolated vrille (vri), a transcription factor previously identified as having a role in development. The VRI protein shows homology to DBP, a transcription factor that oscillates in the liver and in the SCN. The expression of vri is synchronous with that of per and tim and is low in flies mutated for Clock, suggesting that vri is regulated by the transcriptional regulatory loops formed by the other clock proteins. A pace-setting trend? A major issue of clock research has been the identification of anatomical structures containing independent
pacemakers. Results in zebrafish presented by N. S. Foulkes and P. SassoneCorsi (Strasbourg, France) indicate that expression of the Clock gene homologue oscillates not only in the two defined clock structures – the eye and pineal gland – but also in peripheral tissues such as the heart and kidney. A similar pattern was also observed for the two zebrafish CLOCK partners, BMAL1 and BMAL2. The finding is even more striking as the oscillation is maintained in organ ex vivo cultures of these tissues. The presence of peripheral oscillators in Drosophila was demonstrated elegantly by P. Hardin (Houston, Texas) in the fly antenna by looking at the circadian response of electrical activity induced by odorant stimuli. If oscillators are present in peripheral tissues, then there is a chance that they might also be revealed in single cells. Experiments conducted by U. Schibler (Geneva, Switzerland) indicate the presence of circadian oscillations in gene expression in cultured immortalized fibroblasts in response to serum. This fibroblastic clock is independent of the cell cycle but is influenced in different ways by agents acting on various intracellular signalling pathways. Time to take stock Our understanding of the functioning of the circadian clock is progressing by leaps and bounds, and the future will undoubtedly hold many surprises. Crucial questions are: • what is the biological role of peripheral clocks? • how does light entrain the clock? • how do the molecular components of the clock work together, and • how are they modulated by intracellular pathways? Hopefully, we will have the answers in a forthcoming meeting.
http://tto.trends.com New articles published recently in Technical Tips Online include: A trilogy of Core Protocols: • Parchaliuk, D. L., Kirkpatrick, R. D., Simon, S. L., Agatep, R. and Gietz, R. D. (1999) Yeast two-hybrid system: part A –screen preparation (p01616); part B – screening procedure (p01713); part C – characterizing positives (p01714) (http://tto.trends.com) and two new Technical Tips: • Kuschak, T. I., Paul, J. T., Wright, J. A., Mushinski, J. F. and Mai, S. (1999) FISH on purified extrachromosomal DNA molecules (http://tto.trends.com) t01669 • Coleman, A. E., Angkustsiri, K. and Janz, S. (1999) Use of B1-repeat-supplemented Cot-1 DNA to enhance background suppression in FISH (http://tto.trends.com) t01769
372
trends in CELL BIOLOGY (Vol. 9) September 1999