Time, Flies

Sehgal’s parents had also joined the visit and were returning to India in July, shortly before she started the PhD program. “It was fortuitous the way things worked out. My parents were comfortable leaving me in New York because my oldest sister was living there.” One month later, however, her sister and family moved to Florida, and Sehgal was alone, living in Cornell housing. “The first six months were really, really rough,” she says. Cornell had dissolved the genetics program to which Sehgal had been admitted and offered her tuition support with no stipend—and that only for the first semester. “My parents and sister were in no position to help me financially,” she says. Sehgal found a professor at the adjacent Memorial Sloan Kettering Cancer Center (MSKCC), Raju Chaganti, who gave her part-time work with no expectation that she join his lab. She had little money and survived on ramen noodles. “Everyone jokes about living on ramen noodles in college, but I literally did. You could buy four to six packets for $1 depending on how good the sale was.” But by the end of her first semester, Sehgal’s situation took a turn for the better. The chair of the cell biology program picked up Sehgal’s tuition and stipend support. By the end of the year, she had joined Moses Chao’s lab and was trying to clone the gene for the human nerve growth factor (NGF) receptor.

“If someone during my postdoc had asked me if flies sleep, I would have said, ‘Sure, why not.’ But no one asked.”

After focusing on neuronal development during her PhD studies, Sehgal switched to studying circadian rhythms in fruit flies as a postdoc. As a professor, she continued to unpack the molecular mechanisms of biological clocks—and since discovering that fruit flies sleep, Sehgal’s laboratory has continued to use Drosophila melanogaster to probe the molecular circuits that govern sleep. Here, Sehgal talks about how, as a graduate student, she set off the emergency shower in the lab; her rash decision to study circadian rhythms; and why she has never generated beautiful data.

SEHGAL SEEKS

On the move. Sehgal was born in New Delhi. Because her father worked in the Indian government’s tourism department, the family moved around while she was growing up, first to Frankfurt, Germany, where Sehgal attended kindergarten and first grade, then to Calcutta, back to Delhi, and on to Kashmir. When she was in ninth grade, Sehgal’s parents sent her to live with relatives in Meerut, a city near Delhi, so she could attend a high school they thought was better than the ones in Kashmir. When she had to select an education track toward the end of high school, she chose a science track because that’s what her friends were doing, but coupled it with English literature, which she most enjoyed.

Uninspired science. Sehgal attended an all-girls’ college at Delhi University, graduating in two years with a degree in biology. The two-year option was available for specific majors because the college system had not yet been synced with India’s new 12-grade high school program. The curriculum was old-fashioned chemistry, botany, and zoology with an emphasis on rote memorization, according to Sehgal. “Part of the reason I was not interested in science was the way it was taught. There was no effort to make it interesting.” After graduating in 1980, Sehgal was accepted to law school. She also applied to a prestigious molecular biology master’s program at Jawaharlal Nehru University in Delhi. To her surprise, she got in and, following the advice of friends and family, chose the master’s program.  

At a crossroads. While Sehgal was completing her master’s, her family moved to Perth, Australia. She joined them after completing the program in 1982 and worked in a laboratory at the Royal Perth Hospital, studying DNA repair in muscular dystrophy patients. But she hated the monotonous cell irradiation and counting. “I was worried that I needed to make a career decision and didn’t know what to do,” says Sehgal. With four years invested in science, she decided to apply to graduate school in the U.S.

What bands? “The first semester was miserable for many reasons,” says Sehgal of her initial time at Cornell. “I didn’t know if I would be there after the semester, and the classes were really hard. I had never read a research paper before. We would discuss bands on gels and I had no idea what anyone was talking about! ‘What is a gel and what is a band?’ I thought.” Sehgal met an older Indian graduate student from Canada. She confided in him how worried she was about failing out. “‘One thing you need to learn about Americans is that they always act like they know everything,’ he said to me. He turned out to be mostly right. After our first exam in biochemistry, I got the second highest score, which was a big shock to me. I thought everyone knew more than me. But it was also a shock to the other students. Five people had failed the exam, and someone asked me if I was one of them.”

SEHGAL STEADIES

Last place. During her rotation in Chao’s lab, she tried to clone the gene for the human NGF receptor (NFGR) by transfecting human DNA into mouse fibroblasts. By the time she moved on to her other rotations, Chao had verified that the human DNA left after her transfections was indeed the NGFR gene. When the gene cloning was published in Science, Chao made Sehgal last author. “I can only guess, but I think he had to be first author because cloning this gene was something he had always wanted to do; it was his project. At the same time, he wanted to give me the next- best authorship,” Sehgal says.

Fly clock. For her postdoc, Sehgal wanted to continue to study neuronal development but switch to Drosophila, a system that was malleable to genetic manipulation. Rather than doing a postdoc in California as she had planned, Sehgal applied to join Michael Young’s lab at Rockefeller University in NYC, as her fiancé and now husband, Jeffrey Field—presently a professor of pharmacology at Penn—had recently been promoted to staff member at the Cold Spring Harbor Laboratory.

During her interview, Young had handed her an accepted manuscript on circadian rhythm, and Sehgal switched to working on biological clocks. Following discussions with Young, she began a genetic screen to identify novel circadian rhythm mutants. “I was very naive. Had I stopped to think whether I should do this screen, the answer would certainly have been ‘no,’ because it had been 20 years since the first clock gene was identified.” Sehgal screened mutations for deviation from the timing of eclosure—the emergence of adult flies from their pupae—which occurs at dawn in wild-type flies. She also had to set up equipment to monitor flies’ rest-activity cycle (used as a readout for circadian rhythm mutants), which required tracking down discontinued Apple 2E computers that could support the analysis software. After about a year of setup, another postdoc, Jeff Price, joined the project, and together they identified timeless, a novel circadian rhythm mutant with alterations in both rest-activity behavior and eclosure timing. The timeless gene—not linked to the mobilized P-elements used to mutagenize the flies—took yet another year to clone, which Sehgal and Young accomplished after she became a Penn faculty member in 1993. In an accompanying paper, Sehgal described how the periodicity of timeless transcription controls the accumulation and nuclear localization of the protein of the other known circadian rhythm gene, period. In 1996, Sehgal’s graduate student Melissa Hunter-Ensor found that the Timeless protein is degraded by light, “providing a mechanism of how the clock is reset daily,” says Sehgal.

Fly sleep. “It was known that flies had a rest-activity cycle, which we used to determine what the internal clock was doing, but sleep was not studied as a behavior in and of itself. If someone during my postdoc had asked me if flies sleep, I would have said, ‘Sure, why not.’ But no one asked,” says Sehgal. It was only after she came to Penn that she began to interact with researchers at the university’s Sleep Center and started to wonder if flies actually did sleep. Joan Hendricks, a Penn School of Veterinary Medicine sleep researcher, came to Sehgal’s lab to learn molecular biology. On the heels of using genetics to identify circadian-rhythm genes, Hendricks and Sehgal wanted to see if they could establish the fruit fly as a sleep model. “Joan basically locked herself in the dark room with a safe light and watched the flies, because we needed to see what the behavior looked like. We decided to forget about EEG tracking in flies and looked for other sleep characteristics: control by a circadian clock, reversibility, an increased arousal threshold to sensory stimulation, and most importantly, the need to make up for loss of the rest state when deprived of it,” says Sehgal.

In 2000, they showed that the flies’ rest-like state is indeed sleep and that human sleep-perturbing drugs can also alter sleep patterns in flies, suggesting similar neurochemistry is at work. “The definition for fly sleep is five minutes or more of inactivity. Less than five minutes and they are still responsive to gentle stimulation,” explains Sehgal. To monitor the flies’ sleep and activity states, her lab continues to use an activity monitoring system similar to the one she set up during her postdoc: researchers place flies individually in small tubes, and assay their movement through deflection of an infrared beam projected into the tube. For sleep deprivation, they attach vials of flies to a shaker that rotates randomly, giving the insects jolts that wake them up.

Sleep and memory. In 2006, postdoc William Joiner homed in on the cAMP/protein kinase A (PKA) pathway as a promoter of fly wakefulness and identified the mushroom body, the part of the fly brain associated with memory, as the region of the brain that regulates sleep. “This supported the idea that sleep may be required for consolidation of memory,” says Sehgal.   

New sleep gene. Kyunghee Koh, another postdoc in the lab, collaborated with Joiner and postdoc Mark Wu on the lab’s first unbiased genetic screen for short-sleeping fly mutants in 2005. They found that inactivation of the sleepless gene in flies results in a greater than 80 percent reduction in sleep and cuts their life span in half. The gene codes for a small molecule enriched in the fly brain that modulates ion channel activity.

SUPER SEHGAL

Blooper reel. “I was really green when I started graduate school. I didn’t know simple things like what ethidium bromide was, and when I did my first library screen, I threw away the nitrocellulose with the bacterial colonies and tried to plate the spacer on the petri dishes. My graduate story is filled with disasters. I started a fire in the lab with the flame that was kept in the hood and the firemen came. Another time, I flooded the lab. I was talking to someone and playing with the emergency shower string that was hanging above me. I gave it too strong a tug and the showers went off. I was jumping to catch the string trying to turn it off. Meanwhile, the guy I was speaking to walked away and came back with a bar of soap and a towel for me as a joke.”

“Sleep also affects organs other than the brain, although the sleep field has been very brain-centric. I am very interested now in whether peripheral organs contribute to sleep.”

Convincing results? “I have really bad lab hands. I have never generated beautiful data. Every experiment I did had to be done a billion times, to convince myself and everyone else that I had a result.”

Beyond the brain. “We don’t know if there are dedicated sleep molecules. All of the genes we find in our screens also regulate neural activity and plasticity and some roles in learning and memory. [The genes are] not like the dedicated biological clock genes. Sleep also affects organs other than the brain, although the sleep field has been very brain-centric. I am very interested now in whether peripheral organs contribute to sleep.”

Getting your ZZZZs. “Since I was a kid, sleep has always been super important to me. My parents never had to make me go to bed. Yet my daughters don’t like to sleep, and my older daughter in particular had problems with sleep when she was little. I tease her that I want to sequence her clock genes because I think she has delayed sleep phase syndrome.”

Homebody. “We still live in the same house that we moved into when we first started at Penn in 1993. After being such a nomad, I never thought I would end up living in the same house for more than 20 years.” 

Greatest Hits

• In Drosophila, identified timeless, the second circadian rhythm clock gene found
• Described how light sets the timing of the Drosophila clock
• Discovered that the fruit fly’s rest phase is actually a form of sleep and established Drosophila as a genetic model of sleep
• Isolated sleep-regulating genes and a site in the Drosophila brain that controls sleep
• Discovered molecules that regulate sleep, such as sleepless; identified the cAMP pathway as a regulator of sleep and identified the mushroom body in the fly brain where the pathway acts