ClockNews#20: Zebrafish, Math-Modelling and SAD
With its relatively small genome and its ability to produce mutations that are analogous to those in humans, the humble zebrafish is already a valuable tool in genetics research. But it just got better: researchers at the University of Houston have developed a transgenic zebrafish that glows according to its biological clock.
The fish, which incorporates a gene responsible for the flash of fireflies, should help scientists better understand the genetic basis of circadian rhythms, the metabolic processes linked to cycles of light and dark. Already, the Houston researchers have used it to study how the circadian clock is kick-started in the developing zebrafish.
"What we've done is inserted a recombinant gene into the germ line of these fish," said Dr. Gregory M. Cahill, an associate professor and the co-author of a paper describing the work with Dr. Maki Kaneko in the open-access journal Public Library of Science Biology.
In their work, the firefly gene is put under the control of a zebrafish gene that is a "biological clock promoter": it controls the expression of the firefly gene according to circadian rhythms. So the firefly gene produces more of an enzyme called luciferase during the morning than the evening.
When zebrafish larvae are put in a special solution, the luciferase causes the larvae to luminesce. This glow, while faint, can be detected by a machine. When measured over time, it reflects the functioning of the fish's internal clock.
With this approach, Dr. Cahill said, large numbers of larvae can be measured at once. "You want to identify mutants where the clock runs too fast or too slow," he said. But mutations are rare, so a lot of zebrafish are needed. "In theory, you can test up to 2,000 animals at a time with this," he said.
Once clock-affecting mutations are discovered, they can be compared with the human genome to better understand the genetic factors behind jet lag or other conditions that affect the body's rhythms. Similar comparative work is being done with fruit flies and mice. But Dr. Cahill said the new technique had some advantages. Unlike fruit flies (and like humans), zebrafish are vertebrates. And they reproduce much more abundantly than mice. "The nice thing about fish is that we can do lots of these, relatively inexpensively," he said.
[Update, added 2-2-2005]:
A much better review:
Glow-in-the-dark zebrafish at UH hold keys to biological clocks
Professor Gregory M. Cahill’s research illuminates a ’first’ in this species Using genetically altered zebrafish that glow in the dark, University of Houston researchers have found new tools that shed light upon biological clock cycles. Gregory M. Cahill, associate professor of biology and biochemistry at UH, and Maki Kaneko, a fellow UH researcher who is now at the University of California-San Diego, presented their findings in a paper titled "Light-dependent Development of Circadian Gene Expression in Transgenic Zebrafish," appearing Feb. 1 in the Public Library of Science’s PLoS Biology, an online journal that, along with PLoS Medical, is committed to making scientific and medical literature a public resource. "By injecting the luc gene that makes fireflies glow into our zebrafish, our bottom-line finding goes back to nature versus nurture," Cahill said. "We found that these per3-luc zebrafish contain something in their genetic makeup that gets their clocks ticking without parental influence, however, we determined that it does take some sort of environmental input for the clock to start. In this case it was exposure to light/dark cycles after the fourth day of development, about the age when the fish start to swim and feed." The researchers used zebrafish (danio rerio) because they yield such a high output of spawn, with hundreds of eggs being laid by each female per week. This gives the scientists a better chance of identifying mutant fish whose biological clocks run fast or slow, providing the ability to trace the specific genes that create the anomaly. Putting UH a bit ahead of other institutions engaged in this type of research, Cahill and his team will be able to analyze more than 2,000 zebrafish per week. The per3-luc zebrafish is the first vertebrate system available for this level of high-throughput measurement. "Because we can test so many zebrafish at a time, the one in a thousand odds of finding a mutant are more easily and efficiently attainable," Cahill said. "Ultimately, this type of research can help with tracing why humans develop such things as sleep disorders or mental illnesses like depression." Per3 is the naturally occurring clock-regulated gene. The protein that it encodes is produced at highest levels near dawn, and when the luc gene is inserted into it, the luciferase protein is produced in a similar way. The result is that these fish glow rhythmically, emitting more light during the day than during the night. The amount of light is below the level of detection by the human eye, but it is easily measured with an instrument called a luminometer. "This has given us the tool we need to find other parts of systems that influence biological clocks," Cahill said. "We are optimistic that this will shed light upon such things as reproduction in other light-dependent animals." These findings have laid the groundwork for further study along these lines. With a team now built, UH graduate students who assisted with this project are now trained to work with Cahill to implement the next steps of this research. Prior to coming to UH in 1994, Cahill was a research assistant professor in the Department of Anatomy and Cell biology at the University of Kansas Medical Center in Kansas City and received his postdoctoral training at Emory University. He received his doctorate in biology and neuroscience from the University of Oregon in Eugene, where he studied the mechanisms of circadian responses to light. He graduated with his bachelor of science from the College of Biological Sciences at the University of Minnesota in Minneapolis/St. Paul. His research interests include molecular, cellular and physiological mechanisms of vertebrate circadian rhythmicity, photoreceptor cell and molecular biology, and neurobiology. He is a member of the Society for Research on Biological Rhythms and the Society for Neuroscience and is currently funded under a $1.2 million National Institutes of Health grant through 2007 as the principal investigator on "Genetic analysis of zebrafish circadian rhythmicity," under which this latest study falls.
I just downloaded the paper and printed it out for slow reading at home. Here is the abstract:
Light-Dependent Development of Circadian Gene Expression in Transgenic Zebrafish
Maki Kaneko1¤ , Gregory M. Cahill1*
1 Department of Biology and Biochemistry, University of Houston, Texas, United States of America
The roles of environmental stimuli in initiation and synchronization of circadian oscillation during development appear to vary among different rhythmic processes. In zebrafish, a variety of rhythms emerge in larvae only after exposure to light-dark (LD) cycles, whereas zebrafish period3 (per3) mRNA has been reported to be rhythmic from day 1 of development in constant conditions. We generated transgenic zebrafish in which expression of the firefly luciferase (luc) gene is driven by the zebrafish per3 promoter. Live larvae from these lines are rhythmically bioluminescent, providing the first vertebrate system for high-throughput measurement of circadian gene expression in vivo. Circadian rhythmicity in constant conditions was observed only after 5–6 d of development, and only if the fish were exposed to LD signals after day 4. Regardless of light exposure, a novel developmental profile was observed, with low expression during the first few days and a rapid increase when active swimming begins. Ambient temperature affected the developmental profile and overall levels of per3 and luc mRNA, as well as the critical days in which LD cycles were needed for robust bioluminescence rhythms. In summary, per3-luc zebrafish has revealed complex interactions among developmental events, light, and temperature in the expression of a clock gene.
Received September 23, 2004; Accepted November 19, 2004; Published February 1, 2005
Copyright: © 2005 Kaneko and Cahill. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abbreviations: BAC, bacterial artificial chromosome; cps, counts per second; DD, constant darkness; Kmr, kanamycin resistance gene; LD, light-dark; luc, luciferase gene; MESA, maximum entropy spectral analysis; per, period gene; qPCR, quantitative PCR; SEM, standard error of the mean
Academic Editor: Ueli Schibler, University of Geneva, Switzerland
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
¤ Current address: Division of Biology, University of California-San Diego, La Jolla, California, United States of America
Citation: Kaneko M, Cahill GM (2005) Light-Dependent Development of Circadian Gene Expression in Transgenic Zebrafish. PLoS Biol 3(2): e34.]
NYU profs finding out what makes the biological clock tick
A pair of NYU researchers have developed a model to chart and explain the biological clock that exists in every cell of the human body. Daniel Forger, an NYU mathematician and biologist, and Charles Peskin, a professor at NYU's Courant Institute of Mathematical Sciences and Center for Neural Science, have developed a mathematical model that explains circadian rhythm, the remarkably prcecise 24-hour biological clock found in cells in the body. If this precision is affected, it may have drastic consequences, Forger said. Jet lag, the habitual urge to sleep at certain times and drug symptoms are all directly affected by the circadian clock's timekeeping. Mutations or pharmaceutical drugs could affect the circadian clock, changing its rhythm, he said. "The circadian clock has also been linked to Alzheimer's and cancer," Forger said. "This model can begin to answer some of these questions." The scientists aim to trump previous models in terms of accuracy, tracking and recording large amounts of molecular data that would otherwise be too much for researchers, Forger said. "It's hard to keep track of the numbers of molecules in a cell over time," he said. "That's what makes this model so great. We can manipulate numbers of molecules." But problems can arise with these rhythms. "Clocks wind down over time," Forger said. "That basically happens in cells, too - the clock within." NYU Assistant Professor of Biology and Neural Science Justin Blau has researched circadian rhythms and recognizes the far-reaching effects of the new model. "As for human implications, it would be interesting to use the model to see how the clock can be reset by light most efficiently - which could help travelers adapt to a new time zone as quickly as possible," Blau said. The researchers validated their results with experimental data regarding concentrations of protein molecules within the cells of mice. "What is great about this model is that it makes clear predictions that can be tested experimentally," Blau said. "So this could be a new dawn for math-biology, where work in math leads to experiments in biology." Forger agreed that the research has great potential for the math and science world. "It's a first step," Forger said. •
Beat The Blues This Winter
"Fall back" will have an incredible impact for millions that are plagued by Seasonal Affective Disorder (SAD), commonly referred to as the "Winter Blues." Research in Ontario suggests that between 2 and 3 percent of the general population may have SAD. Another 15 percent have a less severe experience described as the "winter blues."
Recent studies suggest that SAD is more common in northern countries, where the winter day is shorter and it is usually characterized by feelings of sadness, anxiety, and lethargy caused by the overproduction of melatonin, a sleep hormone produced by the brain. Symptoms may also include irritability, cravings for sweet or starchy foods, and significant weight gains.
Weather often affects your mood. Some people, however, are vulnerable to a type of depression that follows a seasonal pattern. For them, the shortening days of late autumn are the beginning of a type of clinical depression that can last until spring. This condition is called "Seasonal Affective Disorder," or SAD.
What Causes SAD?
Research into the causes of SAD is ongoing. However, SAD is thought to be related to seasonal variations in light. A "biological internal clock" in the brain regulates our circadian (daily) rhythms and the production of neurotransmitters that regulate sleep, mood, and appetite.
For many thousands of years, the cycle of human life revolved around the daily cycle of light and dark. We were alert when the sun was up; we slept when our world was in darkness. The introduction of electricity has relieved us of the need to be active mostly in the daylight hours, but our biological clocks may still be telling our bodies to sleep as the days shorten. This puts us out of step with our daily schedules.
What are the Symptoms?
SAD can be difficult to diagnose, since many of the symptoms are similar to those of other types of depression or bipolar disorder. Generally, symptoms that recur during the winter may indicate the presence of SAD. They may include:
Change in appetiteCraving for sweet or starchy foodsWeight gainDecreased energyFatigueTendency to oversleepDifficulty concentratingIrritabilityFeelings of anxiety and despair
The symptoms of SAD generally disappear when spring arrives. For some people, this happens suddenly with a short time of heightened activity. For others, the effects of SAD gradually dissipate.
How is SAD Treated?
If you feel depressed for long periods during autumn and winter, if your sleep and appetite patterns change dramatically and you find yourself thinking about suicide, you should seek professional help. There is effective treatment for SAD. Even people with severe symptoms can get rapid relief once they begin treatment.
People with mild symptoms can benefit from spending more time outdoors during the day and by arranging their environments so that they receive maximum sunlight. Keep curtains open during the day. Move furniture so that you sit near a window. Installing skylights and adding lamps can also help.
Exercise relieves stress, builds energy and increases your mental and physical well-being. Build physical activity into your lifestyle before SAD symptoms take hold. Make a habit of taking a daily noon-hour walk. The activity and increased exposure to natural light can raise your spirits.
Many people with SAD respond well to exposure to bright, artificial light. "Phototherapy," or light therapy, involves sitting under a special fluorescent light box once or twice a day. A health care professional should be consulted before beginning this kind of treatment.
Exposure to bright light stimulates the pineal gland, which suppresses the secretion of melatonin, the sleep hormone commonly overproduced by SAD sufferers. A high fidelity light source of 10,000 LUX, such as the Verilux HappyLite Sunshine Simulator works by providing daylight balanced, soothing, glare-free light in a concentrated "dose."
Increasing your exposure to light, monitoring your diet, sleep patterns and exercise levels are important first steps in maintaining your health and regulating your Circadian Rhythms. For those who are severely affected by SAD, devising a treatment plan with a health care professional consisting of light therapy, medication and/or cognitive-behavioral therapy may help to relieve these depressive symptoms.