Jan's lab : 这里有弟子么？# Faculty - 发考题
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Download pdf published by SfN (Chapter from 'The History of Neuroscience in
Autobiography, Volume 8' edited by Larry R. Squire)
Yuh-Nung Jan's CV
Lily Jan's CV
Yuh-Nung Jan and Lily Jan
Birth
Family History and Growing Up
National Taiwan University
The Hiking Trip to Shitou in the Spring of 1967
Graduate School Application
Graduate Study at Caltech (1968锟974)
Seymour Benzer Lab (1974锟977)
Steve Kuffler锟絪 Lab at Harvard Medical School
and Life in Boston and Woods Hole (1977锟979)
University of California, San Francisco (1979锟絧resent)
Breakthroughs in 1987
Our Family and Life Outside of the Lab
Some Reflections
References
Lily and Yuh-Nung Jan went to Caltech in 1968 after their undergraduate
study in physics at National Taiwan University. After two years of graduate
study in physics, they switched to biology under the influence of Max Delbr
眉ck. They stayed at Caltech for postdoctoral training with Seymour Benzer
and then worked in Steve Kuffler鈥檚 lab at Harvard Medical School to
demonstrate that peptides can function as neurotransmitters. During their
postdoctoral training with Seymour Benzer, they began their long-term
collaboration. Cloning of the first potassium channel gene Shaker and its
mammalian homolog in the Jan lab at University of California, San Francisco
(UCSF), followed up with expression cloning of a founding member of the
inwardly rectifying potassium channels and the founding member of a novel
calcium-activated chloride channel family, has led to molecular and cell
biological studies of how these ion channels work and how they contribute to
neuronal signaling. In parallel to these ion channel studies, the Jans
started their work on neural development at UCSF in order to understand how
neurons acquire their specific cell fate and morphology. Their discoveries
include atonal鈥攁 founding member of the large family of proneural genes
that endow cells with neuronal cell fates, and numb鈥攖he first cell fate
determinant exhibiting asymmetric localization in dividing neural precursor
cells. More recently, they have begun unraveling the logic and underlying
mechanisms for generating diversity in neuronal morphology (especially
dendritic morphology) and learning how such diversity contributes to the
wiring of the nervous system
Yuh-Nung Jan
Born:
Shanghai, China
December 20, 1946
Education:
National Taiwan University, BS (1967)
California Institute of Technology, PhD (1974)
Appointments:
Postdoctoral Research Fellow, California Institute of Technology (1974)
Postdoctoral Research Fellow, Harvard Medical School (1977)
Assistant Professor, University of California, San Francisco (1979)
Investigator, Howard Hughes Medical Institute (1984)
Honors and Awards (Selected):
McKnight Scholar Award (1978鈥981)
Elected member, National Academy of Sciences (1996)
Elected member, Academia Sinica, Taiwan (1998)
Distinguished Alumni Award, California Institute of Technology (2006)
Elected member, American Academy of Arts and Sciences (2007)
Javits Neuroscience Investigator Award, National Institute of
Neurological Disorders and Stroke,
National Institutes of Health (2010)
Seymour Benzer Lecture, Neurobiology of Drosophila meeting, Cold Spring
Harbor Lab (2011)
Honors and Awards Shared by Lily Jan and Yuh-Nung Jan:
W. Alden Spencer Award and Lectureship, Columbia University (1988)
38th Faculty Lecturer Award, University of California, San Francisco (
1995)
Harvey Lecture, New York (1998)
The Stephen W. Kuffler Lecture, Harvard Medical School (1999)
K. S. Cole Award, Biophysical Society (2004)
Jan Lab Symposium (2006)
Society of Chinese Bioscientists in America Presidential Award (2006)
Ralph Gerard Prize, Society for Neuroscience (2009)
Edward M. Scolnick Prize in Neuroscience, Massachusetts Institute of
Technology (2010)
Albert and Ellen Grass Lecture, Society for Neuroscience (2010)
Wiley Prize in Biomedical Sciences (2011)
Gruber Neuroscience Prize (2012)
Lily Jan
Born:
Fu-Chow, China
January 20, 1947
Education:
National Taiwan University, BS (1968)
California Institute of Technology, PhD (1974)
Appointments:
Postdoctoral Research Fellow, California Institute of Technology (1974)
Postdoctoral Research Fellow, Harvard Medical School (1977)
Assistant Professor, University of California, San Francisco (1979)
Investigator, Howard Hughes Medical Institute (1984)
Honors and Awards (Selected):
Alfred P. Sloan Research Fellowship (1977锟979)鈥br />
Klingenstein
Fellowship Award (1983锟986)
Elected member, National Academy of Sciences (1996)
Elected member, Academia Sinica, Taiwan (1998)
Distinguished Alumni Award, California Institute of Technology (2006)
National Institute of Mental Health MERIT Award (2006)
Elected member, American Academy of Arts and Sciences (2007)
Honors and Awards Shared by Lily Jan and Yuh-Nung Jan:
W. Alden Spencer Award and Lectureship, Columbia University (1988)
38th Faculty Lecturer Award, University of California, San Francisco (
1995)
Harvey Lecture, New York (1998)
The Stephen W. Kuffler Lecture, Harvard Medical School (1999)
K. S. Cole Award, Biophysical Society (2004)
Jan Lab Symposium (2006)
Society of Chinese Bioscientists in America Presidential Award (2006)
Ralph Gerard Prize, Society for Neuroscience (2009)
Edward M. Scolnick Prize in Neuroscience, Massachusetts Institute of
Technology (2010)
Albert and Ellen Grass Lecture, Society for Neuroscience (2010)
Wiley Prize in Biomedical Sciences (2011)
Gruber Neuroscience Prize (2012)
Yuh-Nung Jan and Lily Jan
Birth
We were born nine days apart. Yuh-Nung锟絪 birthday was listed as December
20, 1946, but that is according to the lunar calendar commonly used then. It
corresponds to January 11, 1947, in the Western calendar, which is nine
days ahead of Lily锟絪 birthday, January 20, 1947. Because Yuh-Nung was born
slightly ahead of his due date, we might have started our embryonic
development around the same time.
Family History and Growing Up
Yuh-Nung鈥檚 father, Ten-Sun Jan, was from a well-to-do family in Jiangxi
Province, China. Originally, Yuh-Nung鈥檚 grandfather planned to send his
son to the United States to study economics. However, the outbreak of the
Sino-Japanese war in 1937 altered the plan. Instead, Yuh-Nung鈥檚 father
finished college in China and then joined the Nationalist army to resist
Japanese invasion. He became an officer during World War II. Yuh-Nung鈥檚
mother, Li-Ju Chen, was from Anhui Province and went to the same college as
his father. They got married toward the end of the war and moved to Shanghai
. Yuh-Nung was born there as their first son.
In 1949, as communists were taking over, Yuh-Nung鈥檚 parents got out of
mainland China at the last minute and escaped to Taiwan. During the first
few years in Taiwan, the family lived in a small rural town of Xinpu in Hsin
-Chu County, about 70 km south of Taipei. At the time, Yuh-Nung鈥檚 father
was still in the military and was stationed in the garrison in the frontline
(Quemoy Island). His mother had to work and needed childcare. She persuaded
the local elementary school to take in Yuh-Nung as a first-grader on a
trial basis even though he was only four and a half years old. Yuh-Nung was
able to keep up academically with the older kids in the class, so the school
let him continue. Later, after his father left the military and started
working for the government, the family moved to Taipei, and Yuh-Nung entered
the Jian-Guo High School, which was (and still is) the top high school for
boys in Taipei (the counterpart of Lily鈥檚 Taipei First Girls鈥High
School).
Initially, Yuh-Nung was an indifferent student at the Jian-Guo High School.
He spent most of his time reading novels, daydreaming, playing sports and
the game of go, and so on and paid little attention to schoolwork. He earned
barely passable grades. In his junior year, something clicked inside him.
Influenced by two superb science teachers (in biology and chemistry) and
several academically strong classmates that were his good friends, he became
very interested in science, especially physics and chemistry. In the last
year of high school, he became very motivated and studied hard in
preparation for attending a university to study science.
At the time, the university admission system in Taiwan had some unusual
features and was quite different from that in the United States. The
students were admitted into a particular university department, so each
student had to decide on their major before entering college. There were two
ways of being admitted to the university and the department of choice. Each
of the 10 or so top high schools throughout Taiwan, including Jian-Guo High
School and Taipei First Girls鈥High School, could send a few students
with the highest grades (the top 1 or 2 percent in those schools) directly
to the universities. (Lily was one of those very few.) The vast majority of
the students took a common entrance exam for all the universities at the
same time. Each student, in a single rank order determined by the results of
that exam, was assigned a department in a university based on his or her
own priority list; if the slots were filled for the top choice on the list,
the department the student would join would be determined collectively by
the priority lists and by the entrance exam scores of all high school
seniors. At that time, National Taiwan University (NTU) was 鈥渢he鈥
university among a dozen or so in Taiwan. Acceptance there was very
competitive, especially for the most desirable departments鈥攕uch as physics
, medicine, electrical engineering, and chemistry鈥攂ecause they would fill
their slots very quickly with the topscoring students. In this system,
except for the few students such as Lily who could bypass the entrance exam
because of stellar high school grades, grades had no bearing whatsoever on
university admission. It depended entirely on the results of this intense,
annual, two-day exam. Yuh-Nung benefited from this system. Despite his so-so
grades, he could choose any department he wanted because he did very well
on that exam. (His score was among the top 10 out of approximately 30,000
students.) He agonized over his final two choices鈥攖he department of
physics and the departmentof medicine at NTU. He could not decide and
resorted to a coin toss. The coin toss was for the department of medicine,
but he picked the department of physics instead. Eventually, however, he
ended up with a career in the school of medicine at University of California
, San Francisco (UCSF). That coin toss was 鈥渄eterministic鈥after all.
Like any traditional Chinese family, Yuh-Nung鈥檚 parents cared very much
about their kids鈥education and future but nevertheless allowed him
complete freedom to choose his path. (Several years later, Yuh-Nung鈥檚 only
sibling, his younger brother, Jonathan, followed his footsteps into the
physics department of NTU.)
To go back a bit, Lily was born shortly after the end of World War II in Fu-
chow, China. Her parents, Hong-Shu Yeh and Chuan-Hwa Lee, both accountants,
brought her to Taiwan as a baby. There, she grew up and was drawn into
science while attending Taipei First Girls鈥High School, a fairly large
school with several dozen students in each of a score of classrooms for the
six grades. All students had neatly trimmed short hair that was not allowed
to extend past the earlobe, and all wore the unusual uniform of green shirts
, black skirts, white socks, and canvas shoes. A subset of students was
assigned to Principal Jiang鈥檚 鈥渆xperimental classrooms鈥濃the Kung (
fairness), Chung (sincerity), Qing (diligence), and Yi (perseverance)
classroom (Ban in Chinese). Lily was in the Qing Ban and studied subjects
such as three-dimensional geometry. Lily was fortunate to have a chemistry
teacher who offered students opportunities such as reading college level
textbooks and working on additional problem sets that he took the time to
grade and for which he provided feedback. So, when faced with the choice of
three tracks for college entrance, Lily chose the track for math and
sciences. When given the opportunity to choose a department, Lily knew very
little about the different departments and made her choice more capriciously
than by tossing a coin. When told by Lily鈥檚 high school classmate of her
elder brother鈥檚 advice about the two departments not suitable for girls鈥
攑hysics and electrical engineering鈥擫ily then chose the physics department.
Around that time, students in Taiwan were influenced by the awarding of the
1957 Nobel Prize in physics to Tsung Dao Lee and Chen Ning Yang for their
theoretical work on nonconservation of parity in weak interactions. Chien-
Shiung Wu, the experimental physicist who demonstrated this parity violation
, made multiple visits to Taiwan to talk to young students in the 1960s.
Lily has vivid memories of Wu鈥檚 lectures and her advice to aspiring
science students to consider biophysics as an emerging field of interest.
Not quite following Wu鈥檚 advice, Lily chose to pursue graduate study in
theoretical high-energy physics when she was an undergraduate student in the
physics department of National Taiwan University (NTU)
National Taiwan University
Yuh-Nung entered the physics department of NTU in 1963, and Lily entered the
same department the following year. At that time, it was mostly a teaching
rather than a research-oriented department. The research facility and the
opportunity to be mentored by top-notch faculty were nowhere near physics
department standards for major research universities in the United States (
although it is much better now). However, the students were an outstanding
group because the department was among the hardest to get in. In general,
students were smart and very motivated, and they formed study groups and
inspired one another. Although lacking the experience in cuttingedge
research, these students did receive a strong foundation in physics and
mathematics, which prepared them well for pursuing advanced training, mostly
in U.S. graduate schools. Many of our classmates went on to have successful
careers in a variety of fields. Perhaps the most notable is Andrew Chi-Chih
Yao. Andrew and Yuh-Nung had lived on the same street in Taipei (their
families lived three houses apart) since their early teens. They were
classmates, friends, and rivals in high school and at the university. Andrew
was exceptionally good at mathematics. After earning his PhD in physics, he
switched to computer science. He was a professor at Stanford and then at
Princeton. In 2000, he received the Turing Award, the highest honor in
computer science. In 2004, he moved to China to direct the Institute for
Theoretical Computer Science at Tsinghua University in Beijing.
The Hiking Trip to Shitou in the Spring of 1967
Because more than 90 percent of the physics students were male and had
mandatory military service the year after college, Lily was with the class
one year ahead of hers when applying for graduate school, and she went along
for their graduation trip as well. At that time, traveling within the
island of Taiwan was still a big deal reserved for special occasions such as
the grand field trip celebrating graduation from college. The destination
of that year鈥檚 graduation trip was Shitou, a beautiful forest recreation
area located in the mountainous region of central Taiwan. During that week
of train rides and hiking in the mountains, we got to know each other. Yuh-
Nung was smitten and began pursuing Lily. Many years later, we participated
not only in one special Wu Chien-Shiung Science Camp but also several Wu Ta
You Science Camps. The latter were week-long camps organized by Academia
Sinica in honor of Wu Ta You, a distinguished physicist who was a former
Academia Sinica president and a mentor to Tsung Dao Lee and Chen Ning Yang.
For this camp, around a hundred or so bright undergraduates (selected from
Taiwan and several countries in Eastern Asia) who were interested in
biomedical or physical sciences spent the week with a number of experienced
scientists and were exposed, in alternating years, to various areas of
modern biology or physics. We participated not only because it was a worthy
cause but also for sentimental reasons because the camps were held in Shitou
, where we first met.
Graduate School Application
During Lily鈥檚 senior year in NTU, Yuh-Nung fulfilled his military service
as a communication and electronics officer in the Taiwanese Air Force
stationed at an air base about 30 km from Taipei. He got a motorcycle so he
could ride to Taipei to see Lily every chance he could sneak out of the base
. One of the things we were doing together was applying for graduate school.
Nowadays, one can pursue advanced studies in Taiwan. Back then, one had to
go abroad (mostly to the United States). Because we both wanted to do
theoretical high-energy physics, Caltech was our dream school. Its physics
department had the towering figures of Richard Feynman and Murray Gell-Mann
as well as a constellation of stellar theoretical and experimental
physicists. In prior years, very few NTU physics students were admitted into
the Caltech physics department. As luck would have it, in 1967, the
department accepted a student, Wei-Dou Ni, from the NTU physics department
in the class one year ahead of Yuh-Nung鈥檚. Wei-Dou performed superbly at
Caltech and that undoubtedly helped the subsequent applications of NTU
physics students. In 1968, the Caltech physics department accepted three
students, Chi-Shin Wang (a brilliant student in Yuh-Nung鈥檚 class) and the
two of us from NTU; this was unprecedented.
Chi-Shin became a very successful entrepreneur in Silicon Valley. After
Caltech, Chi-Shin got a degree in electrical engineering at Stanford. After
working for Hewlett Packard for a few years, he started his own company in
Silicon Valley. Among his successes were his pioneering commercial
applications of the global positioning system (GPS).
Graduate Study at Caltech (1968锟974)
Physics Department (1968鈥970)
In September 1968, we arrived in Pasadena from Taipei (see Figure 1). For
this first trip overseas, we brought all our belongings in two suitcases
that could be carried. Caltech is an academic institution with a superb
student to faculty ratio of about four to one and a very small but highly
selective student body. In 1968, most of the 750 undergrads and 750 graduate
students had nice housing on campus. Yuh-Nung stayed in one of the
buildings for male graduate students. However, with no female undergraduate
students and only a very few female graduate students, in 1968, the Caltech
faculty had just created a graduate women鈥檚 house. In the corner house at
293 South Chester Street, Caltech converted seven rooms into bedrooms and
there Lily joined six other female graduate students. She stayed in this
brand new dorm for three years. To satisfy the fire code for a dorm, which
required multiple fire exits for the two upstairs bedrooms, Lily was handed
some ropes and told to throw the ropes out of her bedroom window in case of
a fire. These ropes were only used once鈥攆or a demonstration; somehow
nothing ever triggered the fire alarm even with the seven students taking
turns cooking dinner every week.
Switch to Biology (1970)
As fellow graduate students in theoretical physics, we became aware of the
excitement exuded by biology graduate students we ran into in the dorms and
in the small Caltech community. Although we went to Caltech for its great
physics department, it happened also to have a fabulous biology division
that gave us our first exposure to modern biology. What triggered the switch
from physics to biology was a speaker invited by Max Delbr眉ck for the
weekly physics seminar in 1970 who introduced the basic concepts of
molecular biology and enzymology, hoping to entice physics students to
consider doing research in biology. When Yuh-Nung went to talk to Max
afterward, Max thought that he wanted to join his lab, and Yuh-Nung thought
why not? He returned with a small flowerpot with a stalk of the fungus
Phycomyces growing in it and became a graduate student in Max鈥檚 lab. Now
that Yuh-Nung had joined Max鈥檚 lab, Lily thought she would find a
different lab for her thesis study, so she knocked on the office doors of
the rest of the biology faculty and asked them to please tell her what their
research was about. What Lily recalls most vividly was the visit with
Jerome Vinograd, an outstanding biophysicist. Instead of telling Lily about
his research, he offered the advice that if she wanted to switch from
physics to biology, 鈥淒on鈥檛 try to apply physics to biology; do what Max
Delbr眉ck did by becoming a biologist and thinking like a biologist.鈥When
Lily joined Max鈥檚 membrane biology subgroup, she began her apprenticeship
by painting lipid bilayers for recording the currents generated by carrier
molecules and got to work in a smog-free room in the sub-basement of the
electrical engineering building. From then on, Max made sure that there was
a total separation of Lily鈥檚 graduate study from Yuh-Nung鈥檚 pursuit of
the sensory transduction processes, including the mysterious avoidance
response of Phycomyces.
In 1971, we were married. It was the time of the Vietnam War, Woodstock, and
general social upheaval in United States. We felt no inclination or
obligation to have a traditional wedding, so we chose the simplest ceremony
possible. It cost just six dollars to get a marriage license and pay for
parking at the Los Angeles courthouse; the mandatory blood tests were free
for students. Two witnesses were needed. With foresight, we asked three
friends to drive to the courthouse to witness the ceremony (in case one got
caught in the Los Angeles traffic). Two made it through the traffic in time
to serve as witnesses when a judge married us in his chamber. The next day,
we celebrated by camping and hiking at Yosemite. We moved out of the
graduate student housing and settled in a little detached cottage on Euclid
Street near Caltech. The rent was only $70 a month.
Having gone through the grueling qualifying exams for physics graduate
students before switching to biology and then barely passing the placement
test on organism biology (so the Caltech biology department did not have to
offer this elementary course for the first time to someone utterly
unprepared but who somehow turned up in the entering class), we joined
fellow biology graduate students in a rebellious challenge to the seven-day,
open-book, open-library written exam customarily administered to students
at the end of their first year. The faculty patiently worked with our class
in multiple meetings to find an alternative qualifying exam format that was
mutually agreeable, and they accepted our suggestion that each student write
a research proposal and then be tested in an oral exam in which the student
would defend his or her own proposal plus a fellow student鈥檚 proposal.
This original format may have been implemented only for the qualifying exams
of our class (students in subsequent years probably thought we were nuts
and came up with their own ideas鈥攑erhaps more reminiscent of the major and
minor proposals).
For this experimental qualifying exam, Lily came up with her own idea for a
proposal to localize rhodopsin in photoreceptors and decided to stick to
this proposal for her thesis study. At a time when raising antibodies for
immunostaining with electron microscopy was a new experimental approach that
entailed purification of not only the protein to be used for immunizing the
rabbit but also the antibody along with the ferritin or hemocyanin to be
conjugated to the antibody, Lily asked Jean Paul Revel, who had just moved
from Harvard to Caltech, to join Max Delbr眉ck as her co-mentors. Then she
promptly disappeared into either Max鈥檚 darkroom in the basement or into
Jean Paul鈥檚 darkroom in the sub-basement for the dissection of chicken
eyes and purification of rhodopsin according to her na茂ve plan. Yuh-Nung
helped out by repeatedly driving Lily to the chicken slaughterhouse to
collect chicken heads in a giant ice chest because Lily did not learn to
drive until much later. Lily鈥檚 fellow graduate students also offered help
with their expertise in rabbit immunization. Somehow the rabbit receiving
rhodopsin injections could tell Lily was nervous and would thump his hind
foot to show his displeasure when he heard her footsteps in the hallway.
Most unfortunately, shortly after the last boosting shot of chicken
rhodopsin, the rabbit displayed the ultimate displeasure by dying of
anaphylactic shock. Despite Jean Paul Revel鈥檚 valiant efforts to salvage
some usable antibody from this rabbit, all those long hours laboring in the
darkroom for the isolation of photoreceptor outer segments from thousands of
chicken eyes and the ensuing rhodopsin purification over the course of one
year came to naught, at a time when Lily was trying to figure out whether
she was cut out for the career path of an experimentalist in biology. The
blessing in disguise turned out to be her switch from chicken eyes to cow
eyes as the source for rhodopsin; rabbit antibody against cow rhodopsin
worked nicely for localizing rhodopsin in mouse photoreceptors鈥攐n the
densely packed discs within as well as on the plasma membrane (Jan and Revel
, 1974).
For Yuh-Nung鈥檚 thesis, he worked on two projects concerning sensory
transduction processes using the fungus Phycomyces. The sporangiophore of
Phycomyces is a giant single cell. It is cylindrical in shape and can reach
several cm in length. It was chosen by Max to study sensory responses
because it can sense light and gravity. Phycomyces displays a mysterious 鈥
渁voidance response.鈥Its growing zone, a segment of the cell several mm
long near the tip of the stalk, can sense the presence of any object placed
a few mm away and grow away from it. This happens in the darkness, and it
does not matter what the object is made of. Yuh-Nung tried hard to figure
out what signal is sensed by Phycomyces, and he was able to rule out just
about anything he could think of. For a while, he and his coworkers thought
that it sensed wind current (Cohen et al., 1975), but that was ruled out
several years later. To this day, the nature of this 鈥渁voidance response鈥
remains a mystery.
The main part of Yuh-Nung鈥檚 thesis was his attempt to begin to understand
the molecular mechanism underlying the sensory transduction processes of
Phycomyces. Because Phycomyces responds to various stimuli by altering the
elongation rate of its cell wall, which is composed of chitin fibers, Yuh-
Nung reasoned that the enzyme chitin synthetase could be an important
readout. He characterized this enzyme biochemically and indeed showed that
blue light regulates the activity of this enzyme (Jan, 1974).
Max Delbr眉ck, Our PhD Thesis Advisor
We were extremely lucky to have Max as our thesis advisor. With his unique
combination of intellect, moral integrity, and charisma, Max was a marvelous
mentor and a great influence on us during our particularly impressionable
stage. One piece of advice from Max was, 鈥淒on鈥檛 do fashionable science.
鈥Max liked to venture into unchartered research areas and detested
entering a field because it was popular or trendy, a trait shared by our
other mentors, Seymour Benzer and Steve Kuffler. Of course, venturing into
terra incognito carries risks. In Max鈥檚 case, his later choice of
Phycomyces to understand sensory transduction was not so successful because
although Phycomyces has the advantage of being a large single cell that has
a rich repertoire of sensory responses, unfortunately, it is an organism not
well suited for genetics or biochemistry. However, his earlier choice of
phage to study the basis of heredity was a great success that helped launch
modern molecular biology.
The virtue of not following the crowd is nicely articulated in this passage
from the biography Genius about Richard Feynman (Gleick, 1992):
鈥淚t will not do you any harm to think in an original fashion.鈥Feynman
said. He offered a probabilistic argument. 鈥淭he odds that your theory will
be in fact right, and that the general thing that everybody鈥檚 working on
will be wrong, is low. But the odds that you, Little Boy Schmidt, will be
the guy who figures a thing out, is not smaller. . . . It鈥檚 very important
that we do not all follow the same fashion. Because it is ninety percent
sure that the answer lies over there, where Gell-Mann is working, what
happens if it doesn鈥檛?鈥br />
鈥淚f you give more money to theoretical physics,鈥he added, 鈥渋t doesn鈥
檛 do any good if it just increases the number of guys following the comet
head. So it is necessary to increase the amount of variety . . . and the
only way to do it is to implore you few guys to take a risk with your lives
that you will never be heard of again, and go off in the wild blue yonder
and see if you can figure it out.鈥br />
Max not only influenced our scientific outlook but also exposed us to many
other things that enriched our lives through regular camping trips and
through gatherings in Delbr眉ck鈥檚 house as part of an extended scientific
family鈥攆or example, camping under the stars in the desert or reading the
works of Samuel Beckett, whose writings Max was very fond of. Max was
delighted that the year he was awarded the Nobel Prize in Physiology or
Medicine with Luria and Hersey, the literature laureate was Samuel Beckett.
Accepted into Seymour Benzer鈥檚 Lab for Postdoc (1974)
In 1973, the thesis work for both of us began to take shape, and it looked
like we might get our PhD degrees in another year or so; it was time for us
to start figuring out what to do next for postdoctoral training.
One day, Yuh-Nung came upon a paper by Yoshiki Hotta and Seymour Benzer, 鈥
淢apping of behavior in Drosophila mosaics鈥published in Nature (Hotta and
Benzer, 1972), in which Hotta and Benzer showed that by making genetic
mosaics and constructing embryonic 鈥渇ate maps鈥it was possible to locate
the anatomical site of abnormalities affecting behavior. It was a very
elegant and interesting paper. It got us interested in the ongoing work in
Benzer鈥檚 lab, and we became very attracted to his approach and to his lab.
Benzer was very picky about accepting people into his lab. Fortunately,
with some persuasion by Max Delbr眉ck, Seymour accepted us.
We then tried to get postdoctoral fellowships to support our work in Benzer
鈥檚 lab, with Yuh-Nung proposing to look for learning mutants and Lily
proposing to fine-tune mosaic fate mapping of the neural circuitry for
visual responses. Unfortunately, the National Institutes of Health (NIH)
deemed that neither was worthy of an NIH postdoctoral fellowship so we had
to look for less conventional funding sources. Lily somehow got the
endorsement of an ophthalmologist from UCLA for her application for a Fight-
for-Sight fellowship. For Yuh-Nung, Benzer said: 鈥淲hy don鈥檛 you apply to
this Scottish Rite Schizophrenia Research Program fellowship?鈥because
Seymour firmly believed that one could use a fly to study just about any
problem in biology including schizophrenia; meanwhile, Yuh-Nung was thinking
, 鈥淕ee, schizophrenia? Fly? How am I going to pull this off?鈥This was
back in the era before cloning (BC) and preceding the realization of the
remarkable conservation of mechanisms underlying many biological processes.
After a while, an idea emerged鈥攖here is a fly mutant called tan, which has
an abnormal electroretinogram (ERG). It is as if the fly is seeing things
and displaying ERG signals when there is nothing to see, and visual
hallucination is a hallmark of schizophrenia. Moreover, tan mutants have
abnormally low levels of dopamine, and abnormalities in biogenic amines were
thought of as a potential cause of schizophrenia.
With a giant leap of faith, Yuh-Nung wrote a proposal including this
sentence: 鈥淭he existence of a link between catecholamine abnormality and a
visual defect that is analogous to hallucination in Drosophila mutant tan
suggests that it might be used as a model system for schizophrenia.鈥He
sent it in and thought they probably would just laugh at the proposal. To
his astonishment, they actually funded his application. His disbelief soon
morphed into delusion: 鈥淚 can鈥檛 believe they bought this, imagine what I
can do with a less outlandish proposal, maybe I have a future in this
business.鈥Shortly after, the bubble burst when Yuh-Nung attended a small
neuroscience meeting. A very prominent neuroscientist saw his nametag and
told him: 鈥淎h, I read your proposal. I don鈥檛 believe a word of it, but
since you are working with Seymour, you鈥檒l do alright.鈥br />
Cold Spring Harbor Summer Courses and the Beginning of Our Collaboration (
1974)
To prepare for our postdoc in the field of neuroscience, we took one lecture
course followed by a lab course in the summer of 1974 at Cold Spring Harbor
(CSH) Laboratory, where we had spent the bulk of our summertime as Max鈥檚
graduate students, with Yuh-Nung involved in the Phycomyces workshop every
summer and Lily doing lab work and taking various summer courses in that
secluded idyllic commune for scientists. Little did we know that those
summer courses in neuroscience would have such a big impact on our research
direction even before we began our postdoctoral study in Seymour鈥檚 lab.
Seymour鈥檚 graduate student Bill Harris was attending the same summer
courses with us, and for the one-week break between courses, the three of us
visited Woods Hole and also visited Doug Kankel, who did his postdoc with
Seymour before joining the Yale faculty. On our way out of Doug鈥檚 lab,
Bill picked up a bottle of fruit flies and handed it to us, so we could get
acquainted with our future experimental animal. That is exactly what we did
for the last three days of the lab course.
Although the lecture course was taught by Mike Dennis, Regis Kelly, Carla
Shatz, and Eric Frank, all hailing from Harvard鈥檚 neurobiology department
鈥攖he only department in neuroscience at that time鈥攖he lab course
instructors came from all over the world and included Jac Sue Kehoe and
Phillip Ascher (from France) and Enrico Stefani (at that time from Mexico).
In that era before the invention of patch-clamp recording, we learned to
record from Aplysia neurons and frog muscles. For the last three days of
free period left to the students to try whatever they would like, we dug out
Drosophila larvae from the mushy food in that old bottle to try out what we
had just learned to do with the frog neuromuscular junction, and we enjoyed
much encouragement and tutoring from our instructors. It turns out the
larval muscles are comparable to frog muscle fibers in diameter but much
shorter, so the muscle response to nerve stimulation or iontophoresis of the
transmitter glutamate could be readily recorded (with our beginner鈥檚 luck
) during this lab exercise.
Those CSH summer courses were a fantastic way to learn neurobiology quickly.
We not only learned a great deal but also made friends with some great
people鈥攐ur teachers and fellow students. One of our fellow students that
we got to know very well was Bob Horvitz. He was on his way to start his
postdoc with Sydney Brenner at the medical research council (MRC) Cambridge.
He would later make tremendous contributions in developmental biology and
especially in programmed cell death, for which he was awarded a Nobel Prize
in 2002. Those CSH summer courses were very intense. We worked 16 hours non-
stop every day and were exhausted by the end of week two of those three-week
-long courses. Bob is the only person we know who took three CSH courses in
a row, an early indication that he would have a great career with his
tremendous energy and intellect.
Seymour Benzer Lab (1974锟977)
Identifying Shaker as a Likely Potassium Channel Gene
In August 1974, we started in Seymour鈥檚 lab. Yuh-Nung began by working on
learning and memory as part of the team that did the initial dunce work (
Dudai et al., 1976). After spending a few months training flies, we thought
that, in order to get to the mechanisms of learning and memory, we needed to
develop some functional assay for synaptic transmission, for example with
electrophysiology. So, we teamed up to put together an electrophysiology rig
in Seymour鈥檚 lab in the fall of 1974 to continue what we started in that
summer at CSH to characterize larval neuromuscular junction (NMJ) and
develop it as an assay for a genetic dissection of synaptic transmission. At
that time, the Journal of Physiology was considered the top journal for
neurobiology, and it was acceptable for us to take the time and circulate
our manuscripts among friends and our CSH summer course instructors until we
felt it was ready for the journal. We took pride in publishing our first
collaborative work (Jan and Jan, 1976a, b).
Assuming we could go through the recording of a couple of preparations in a
few hours, we developed a routine for screening the larvae of behavioral
mutants in Seymour鈥檚 collection to look for abnormal neuromuscular
transmission. Very soon after we started this project, on April 28, 1975, we
recorded from a male ShakerKS133 mutant larva and found it displayed an
extremely large 鈥渦nit size鈥for the excitatory junctional potential in
low calcium Ringer鈥攚hereas normally each nerve stimulation induced either
no response (a failure) or a unit response of the same size as the miniature
excitatory junctional potential. Stimulation of the motor nerve of this
Shaker mutant generated large responses every time (see Figure 2). In the
summer of 1975, the CSH symposium happened to feature the synapse. After
Seymour gave a talk and showed our findings based on recordings using a
dissecting microscope. Mike Dennis, who taught us in the lecture course the
previous summer, offered to teach us to do recordings using a compound
microscope with Nomarski optics that would allow us to see the nerve
terminals and to do extracellular recordings to test whether the mutant
phenotype could be attributed to the nerve or to the muscle of Shaker mutant
larvae. We happily made multiple drives to San Francisco. Each time, we
stayed for a couple weeks in the Mariana鈥檚 guesthouse across the street
from the UCSF campus on Parnassus so that we could spend as much time as
possible in Mike鈥檚 lab in the physiology department. Working closely with
Mike, we could see that the Shaker mutant nerve responded to a single
stimulation with multiple recurrent action potentials so that calcium
iontophoresis several milliseconds after the nerve stimulation could still
induce transmitter release and muscle response.
At this point, we wrote up a paper with Mike with the conclusion that the
abnormality of the Shaker mutant nerve terminal could be a defect in the
potassium channel or in the calcium channel and submitted it to Nature.
While this paper was under review, we did more experiments and found that
the Shaker mutant phenotype could be phenocopied by applying the potassium
channel blocker 4-aminopyridine to wild-type larval preparations. When the
Nature editor informed us that the reviewers鈥comments were favorable but
that we needed to shorten our manuscript significantly, we told the editor
that with these additional experiments we would have to lengthen our paper
instead. So we withdrew it from Nature and submitted our paper to
Proceedings of Royal Society because Seymour was just elected as a foreign
member, and we thought he would get a kick out of communicating our paper to
the Royal Society journal (Jan et al., 1977). Again, the reviewers鈥
comments were favorable, and this time one reviewer asked the editor to
reveal his identity鈥擲ir Bernard Katz鈥攁nd to pass on a number of follow-
up questions that he was curious about. After we did a series of experiments
to address these interesting questions, we wrote a long letter to Katz and
included those results in that letter. Though we never got around to
publishing those studies, we included one set of results in the 1997 review
article in Journal of Physiology (Jan and Jan, 1997).
While we worked with Mike Dennis on Shaker mutant recordings, his colleague
John Heuser was working very hard next door looking for some physical
evidence of exocytosis at the motor nerve ending. Using a fancy machine
custom-made at UCSF to strap a frog nerve muscle preparation around a
suspended piston, which was triggered to slam onto a cold slab chilled with
liquid helium shortly after the delivery of a nerve stimulus, John would
recover that flattened frog muscle for freeze fracture and then disappear
into the electron microscope room to search for omega-shaped contours at the
end plate that had to be very close to the muscle surface to have been
frozen immediately on impact with the cold slab. In one of those neighborly
chats with Mike and John, we wondered whether the Shaker mutant larvae with
prolonged transmitter release could make the task easier to accomplish. When
it became evident that the geometry of the larval nerve terminal鈥攁 large
bouton rather than the nicely elongated end plate of the frog motor nerve
ending鈥攚as not amenable to freeze fracture, we turned to the alternative
approach of treating the frog nerve-muscle preparation with 4-aminopyridine.
Indeed, this treatment also prolonged transmitter release from the frog
motor nerve endings making it rather easy to capture vesicle fusions using
John鈥檚 machine (Heuser et al., 1979). Elated with this success, we had a
very memorable celebratory dinner outing with John and Mike at the trendy
restaurant Caf茅 Sports.
Life in the Benzer Lab
The three years we spent in Seymour鈥檚 lab were a wonderful experience鈥攖
remendously enjoyable and intellectually stimulating. Seymour had a great
sense of humor and an immense curiosity. He was a really fun and influential
person to be with. He attracted a very talented and somewhat eccentric
group of people鈥攆ellow postdocs Chip Quinn, Alain Ghysen, Ilan Deak, and
Yadin Dudai, and Seymour鈥檚 graduate students Bill Harris, Don Ready, and
Duncan Byers were there when we joined the group. Several became lifelong
friends, especially Alain Ghysen, who had a strong influence on our later
scientific direction. Several important lines of research were initiated in
Seymour鈥檚 lab during that period. For example, Don Ready laid the
groundwork for Drosophila eye development (Ready et al., 1976), and Bill
Harris discovered the sevenless mutant (Harris et al., 1976), which led to
insights about the mechanisms of induction in retinal cell fate
specification.
Before we had our first child, toward the end of our stay in Seymour鈥檚 lab
, we were 鈥渙wls鈥with respect to our circadian rhythm. Each weekday, we
would get up just before noon and go to Seymour鈥檚 cramped lunchroom to eat
with the whole group. There were lively and free-flowing conversations and
gossip about science, movies, and often food, a favorite subject of Seymour
鈥檚. Those lunches could last for hours. Seymour was a good friend of many
prominent scientists, and from time to time they joined in those lunches as
well. One of the most memorable was the time when Richard Feynman came over
from the physics department. He asked us what we were doing with our
Drosophila learning studies. In a couple of hours, he managed to think of
every clever experiment that had taken several of us months to come up with.
His mind was really impressive.
In the afternoon, after those long lunches, we started our daily work or
went to seminars/lectures. After dinner, we came back to the lab and often
stayed till 2鈥 a.m. We could work uninterrupted for six or seven hours
and that was when we did most of our experiments. Another night owl was Ed
Lewis. He had an even more extreme schedule. He often came back to the lab
around midnight and worked till dawn. That was the period when Ed made a
breakthrough in his studies of the bithorax complex by using the embryo
cuticles to analyze the various chromosome deficiencies and mutants of the
bithorax complex; this made it possible to decipher the effect of lethal
mutations on body patterning and to expand the analyses beyond previous
studies done with adult flies (Lewis, 1978). On several occasions, Ed was
very excited by his new findings and wanted to show someone; we were often
the only ones around. We got a glimpse of his progress and shared his
excitement. Because we were then studying the larval neuromuscular
preparation, we even started a little collaboration with Ed to see how the
internal tissues might be altered in the bithorax mutants. As with much of
Ed鈥檚 work, that was never published. Nevertheless, it was a privilege to
get to know Ed. Years later, when Ed organized a symposium in honor of
Seymour鈥檚 70th birthday in 1991, it was a wonderful opportunity to meet up
with Seymour鈥檚 old friends and disciples (see Figure 3).
What to Do Next?
Having gone into the field of neurogenetics with the hope that genetics
could help with the identification of key genes for neural signaling in much
the same way as it did for biosynthetic pathways, we were hopeful that
molecular biological approaches being pioneered by David Hogness and
colleagues for positional cloning of Drosophila genes might apply to Shaker
cloning with the potential of molecular identification of a potassium
channel without having to take on the daunting task of purifying potassium
channels. However, cloning was in its infancy, and without any training in
molecular biology we were not ready to take on such a challenging task. We
were wondering what to do next. Mike Dennis had spent some time previously
as a postdoc with Steve Kuffler at Harvard Medical School (HMS) and thought
that it might be a good idea for us to go there to work with Steve to gain
more experience in neurophysiology. At that time, the department of
neurobiology at HMS was 鈥渢he鈥neurobiology department in the country.
Steve was a towering figure in neurobiology, and there were many really
outstanding people in that department. Seymour also endorsed this idea
because Seymour was a good friend of Steve鈥檚 and many other people in that
department, and he had a very high regard for them. Around that time, an
Ivy League school asked whether we might be interested in applying for a
faculty position. We agonized over whether to start applying for faculty
jobs or to do a second postdoc at HMS. We spent a long afternoon walking and
talking in the beautiful garden of the Huntington Library near Caltech to
try to figure out what to do. Finally, we decided that getting additional
postdoctoral training with Steve and spending some time in that great
department could only benefit us in the long run.
Birth of Emily and the Move to Boston
The last months at Caltech before we moved to Boston were a very hectic time
for us. Our daughter Emily was born on August 6, 1977, a couple of weeks
ahead of her due date, and we were not quite prepared. On August 5, we were
at a group meeting in Seymour鈥檚 lab. Lily started feeling contractions and
glanced at her watch to time them鈥攖hey were not exactly spaced with five-
minute intervals. As the intervals between contractions got shorter, we were
frantically trying to reach the Lamaze instructor for the class we had been
taking in preparation for our child鈥檚 birth in order to get the course
certificate that Yuh-Nung needed in order for him to be allowed in the
delivery room to serve as the Lamaze coach. As we were driving to the Kaiser
hospital in Hollywood, we were trying to come up with a name for the new
baby, one for a boy and one for a girl, because we did not know the sex of
the baby. After a long night of labor, a wonderful girl, Emily Huan-Ching
Jan, was born early the next morning.
Finally, it was time to leave Pasadena. In September 1977, we packed up and
drove across country to Boston with seven-week-old Emily. Pasadena and
Caltech were our home for nine years, and we spent our entire twenties there
. We had arrived in California very na茂ve鈥攃ulturally and scientifically鈥
and we grew up there. Caltech influenced us more than anywhere else. It
was a great privilege for us to know the tremendous scientists and human
beings Max Delbr眉ck, Seymour Benzer, Ed Lewis, and their wonderful families
and many other wonderful people at Caltech. In 2006, we both were given the
Distinguished Alumni Award at Caltech, one of our most treasured awards, as
if our alma mater was telling us 鈥測ou kids did all right,鈥akin to
parental approval.
Steve Kuffler锟絪 Lab at Harvard Medical School and Life in Boston and Woods
Hole (1977锟979)
Peptidergic Transmission for the Late Slow Excitatory Postsynaptic Potential
In late September 1977, we arrived in Boston to join Steve Kuffler鈥檚 lab.
We were very fortunate to find a very nice flat near Coolidge Corner in
Brookline that was a half-hour walk from the lab. Lily鈥檚 mother flew from
Pasadena to Boston to live with us and helped take care of Emily during the
daytime. We began our new routine of taking shifts to cover the long hours
in the lab and to tend to a baby at home in the evening. Lily would set up
the bullfrog sympathetic ganglia for recording early in the morning and work
together with Yuh-Nung during the day. Yuh-Nung would walk Lily home at
dinnertime to relieve her mother of babysitting and then return to the lab
to resume the experiments.
At that time, Steve was interested in slow synaptic potentials. Initially,
he assigned us to map by electrophysiology the distribution of muscarinic
receptors, which mediate the slow excitatory postsynaptic potential that
lasts for seconds. It was a useful learning experience but was not that
interesting a problem. We became restless after six months and discussed
starting a new project with Steve. We were attracted by the mysterious late
slow excitatory postsynaptic potential (EPSP) lasting for several minutes.
The late slow EPSP, which was initially discovered by Nishi and Koketsu,
persisted in the presence of antagonists for nicotinic acetylcholine
receptors responsible for the fast EPSP and muscarinic acetylcholine
receptors responsible for the slow EPSP. Therefore, some unidentified
transmitter other than acetylcholine has to induce the late slow EPSP. With
Steve鈥檚 blessing, we decided to try to identify the transmitter for the
late slow EPSP, a very interesting but highly risky project.
To try to identify this mysterious transmitter, in May 1978, we began to
apply all kinds of receptor agonists and antagonists to look for an effect
on the late slow EPSP. Besides compounds ordered from Sigma and other
companies, we found some small vials of peptides in the freezer that were
gifts to Steve from Wylie Vale and Jean Revier at the Salk Institute.
Perhaps Steve got these peptides because at the time neuroscientists had
begun to suspect that peptides might function as neurotransmitters. To save
time, we pooled contents from three vials at a time for a quick survey. One
combination produced a moderately encouraging response. Testing them
individually identified the culprit as 鈥淟HRH.鈥We then looked up to see
what LHRH stood for鈥攍utenizing hormone releasing hormone鈥攁nd learned
about the existence of LHRH analogs as potent agonists and antagonists. When
we put those analogs on and observed that the agonist could induce
responses that mimic the late slow EPSP, and the potent antagonist could
block both the LHRH-induced slow depolarization and the nerve-evoked late
slow EPSP, we knew that we were probably on the right track in proposing
that an LHRH-like peptide could be the transmitter mediating the late slow
EPSP. With the new finding, the obvious next step was to demonstrate that an
LHRH-like peptide was indeed present in the presynaptic nerve that
innervates the bullfrog sympathetic ganglia and could be released under
conditions that could elicit the late slow EPSP. The method of choice was
radioimmunoassay, with which we had no experience. We were then told by our
colleagues that there was this new postdoc, Tom Jessell, who had just joined
Gerry Fishbach鈥檚 lab at HMS and who was an expert because he had worked
on substance P for his PhD thesis in the UK. We went to see Tom, and he gave
us many helpful hints.
Then it was time for the Kuffler lab to make its customary migration to
Woods Hole for the summer. Originally, the plan was for Steve and for us to
work primarily on the electrophysiology of the late slow EPSP, but we ended
up doing mostly radioimmunoassays. Steve鈥檚 lab at Woods Hole had great
electrophysiology set-ups but hardly any biochemistry equipment. However,
Woods Hole was such a communal place that we were able to rely on the
kindness of the neighbors to do the necessary experiments.
On the top floor of the Woods Hole lab building with an ocean view on one
side, Steve鈥檚 lab had windows overlooking the tennis courts on the
backside鈥攕urely not a coincidence given that Steve was an enthusiastic
tennis player. Beyond the tennis courts was the apartment for us, a
wonderful arrangement for our daughter and her grandmother to be close by
and for us to slip in some tennis during incubation times for
radioimmunoassays to detect the LHRH-like peptide in bullfrog sympathetic
ganglia and to document the release of the peptide upon nerve stimulation.
We had a wonderful three month stay at Woods Hole and went through the list
of criteria that a putative transmitter must satisfy and ticked them off one
by one. We also got to celebrate our daughter鈥檚 first birthday and go to
the beach for swimming and parties at that vibrant and happy community with
a long history as the favorite summer retreat for biologists. One of Steve鈥
檚 visitors was his old friend and coworker Sir Bernard Katz. That was our
first chance to meet him. We chatted while pushing Emily in a stroller and
watching Steve display his prowess as a tennis player. We were delighted
that he remembered reviewing our Shaker manuscript (Jan et al., 1977) and
that he liked the paper.
By the time the summer was over and we all moved back to Boston, we had
pretty much established that an LHRH-like peptide is the transmitter that
mediates the late slow EPSP. It took only about six months since we started
the project. It was mostly dumb luck. We wrote up the paper with Steve, and
he communicated it to Proceedings of the National Academy of Sciences (PNAS)
(Jan et al., 1979). That work most likely helped several schools become
interested in us and encouraged us to apply for faculty positions. We felt
it was perhaps time for us to get independent positions and to start our own
lab.
Although we spent less than two years in Boston, the experience at the HMS
was very valuable. The neurobiology department was relatively small but had
a very high concentration of terrific neuroscientists. The senior faculty
members were Steve and the other founding members of the department, who had
all worked with him in their youth鈥擠avid Hubel, Torsten Wiesel, Ed
Furshpan, David Potter, and Ed Kravitz. The junior faculty members were Paul
Patterson, Story Landis, Simon Levay, Peter Maclish, Ann Stuart, and John
Hildebrand. Each group typically had only a few highly selected postdocs and
students. When we arrived in 1977, Lou Reichardt, Carla Shatz, and Josh
Sanes had just left. Our contemporaries included among others: Mike Stryker,
Bill Harris, Eric Frank, Doju Yoshikami, Larry Marshall, Bob Stickgold,
Marge Livingstone, Alison Doupe, Mary Kennedy, Charlie Gilbert, and Terry
Sejnowski. It is remarkable how much impact that relatively small group of
people has had on neuroscience.
The department then was very tightly knit. Two core activities were
especially educational. Because all members of the department could fit into
a modest-sized lunchroom, we all had lunch together every day and often had
lunch seminars given by visitors. It was a tough crowd. The speakers were
constantly interrupted, and there was an element of 鈥渨ho could ask the
most critical question鈥during those seminars. We remembered that during a
Christmas party when we were there, 鈥渕ock awards鈥were given out at a
skit. One was given to the person 鈥渨ho most consistently anticipated the
next slide with questioning during those lunch seminars in the previous year
.鈥br />
One of the best department activities was the 鈥渆vening meetings.鈥Every
month or two, the department had dinner together, and one group presented
their ongoing work. Everyone took those presentations seriously because
there was tremendous peer pressure to do well. During those evening meetings
, we learned about much of the exciting work going on in the department.
Job Offer at University of California, San Francisco
After a few months of job interviews, we had several nice job offers and one
was from UCSF. At that time UCSF was not yet a prominent place (like it is
now), and we had better offers in terms of space and start-up funding from
more prestigious places. At UCSF, we had to share a faculty teaching
equivalent (FTE). The lab space and the setup money were very modest as
stated in the job offer letter written by the chair of the physiology
department, Fran Ganong, on December 6, 1978: 鈥Zach Hall and I have
combined our resources to provide 1,000 square feet of space.鈥(That was
for two of us.) And then he wrote: 鈥淚 can also commit $15,000 start-up
money for each of you at this time鈥($30,000 total). Although a dollar in
1978 is worth about three dollars now, $30,000 is not a lot of start-up
money. Nevertheless, we were very attracted to UCSF, especially by the
people there. We already knew Mike Dennis and John Heuser from our previous
collaborations. Zach Hall was recruited from HMS to UCSF to start the
neuroscience program in 1976, and in 1977 and 1978, he recruited Lou
Reichardt and Mike Stryker (both star postdocs) from the neurobiology
department at HMS. Additionally, several other young faculty were already
there including Roger Nicoll, Regis Kelly, Howard Field, Mike Merzenich, and
Allan Basbaum. These young faculty members formed a core that soon
developed into one of the country鈥檚 leading neuroscience programs. Our
other job possibilities were also at excellent places, and there is no way
to know how things might have turned out if we went to one of those instead.
Nevertheless, we felt very fortunate to have decided to come to UCSF to
join this group, and this was one of the best decisions we have ever made.
An unexpected benefit of coming to UCSF that we were not aware of initially
was that, between 1976 and 1979, in parallel to the developing neuroscience
program, a fabulous biochemistry and biophysics department was being greatly
enhanced with the recruitments of these few years: Bruce Alberts, Marc
Kirschner, Keith Yamamoto, Christine Guthrie, Pat O鈥橣arrell, and Tom
Kornberg. Because molecular biology was beginning to revolutionize
neuroscience in the early 1980s, we soon began benefiting from having the
opportunity to interact with those outstanding molecular biologists and cell
biologists.
University of California, San Francisco (1979锟絧resent)
Setting Up Our Modest Little Lab
In late June 1979, we drove across the country during the height of the oil
crisis, with our daughter a toddler not quite two years old, to start our
lab at UCSF on July 1. It took us a couple of months to set up our little
lab on the eighth floor of Health Sciences East (HSE) on the Parnassus
campus (see Figure 4) and then we started doing experiments.
Peptide Acting at a Distance
Initially, we continued with our work on peptides as neurotransmitters. We
discovered that although the LHRH-like peptide was released together with a
classical transmitter, acetylcholine (ACh), from the same nerve terminals
that synapse onto the C type neurons in the sympathetic ganglion, the
peptide can diffuse over tens of microns to act on their true targets (i.e.,
nearby B-type neurons with which the LHRH-like peptide containing
preganglionic nerve fiber does not form a synapse). So the wiring diagram
based on anatomically defined synapses is actually misleading for
identifying the real target of the peptide transmitter (Jan and Jan, 1982b;
Jan et al., 1980). This is something for the connectome folks to consider.
Starting New Projects: Shaker Cloning and Neural Development Studies (1980)
We might have been recruited to the UCSF faculty as electrophysiologists
studying the vertebrate autonomic nervous systems; however, once our peptide
work got going, we soon started switching back to studies of the fruit fly
Drosophila because of the opportunities offered by new developments in the
field. In our youthful exuberance, we initiated two new projects for which
we had no relevant expertise whatsoever: neural development and Shaker
cloning. Looking back, it seemed rather foolhardy to start such risky
projects as beginning assistant professors. Perhaps one reason that we chose
to pursue high-risk projects that interested us was because we felt that we
had the good will and strong support from our chair, Fran Ganong, and the
neuroscience program director, Zach Hall. Indeed, we were tenured rather
quickly (in 1983) even though, by that time, our new projects had yielded
very little concrete results.
Neural Development
Neural development was a question that had been in the back of our minds for
some years. Back in the days when we were in Seymour Benzer鈥檚 lab, in one
of those long lunch gatherings together with fellow postdocs Alain Ghysen,
Ilan Deak, and Yadin Dudai and Seymour鈥檚 graduate students Bill Harris,
Don Ready, and Duncan Byers, we all unabashedly went to the blackboard in
Seymour鈥檚 lunchroom to write down the big questions in neuroscience that
interested us鈥攁 record of those early musings was kept because Seymour
took a Polaroid picture of the scribble on the blackboard. How the nervous
system forms was one obvious question on the list though it was not clear
how one could go about approaching this question.
While in Seymour鈥檚 lab, we became very good friends with a fellow postdoc,
Alain Ghysen. We thought it would be fun to do some work together at some
point. After we set up our little lab at UCSF, Alain and his long-term
collaborator Christine Dambly-Chaudiere would come over from their lab in
Brussels and work together with us to explore all kinds of crazy ideas for a
few weeks at a time every year in the early 1980s. We wanted to work
together on something interesting that had not been worked on by any of us
already. We chose neural development.
Two papers that appeared around that time suggested an approach to study
neural development. The 1980 paper by N眉sslein-Volhard and Wieschaus on 鈥
淢utations affecting the segment number and polarity in Drosophila鈥(N眉
sslein-Volhard and Wieschaus, 1980) was inspiring because it revealed the
involvement of just a score of genes for the specification of the body plan
and demonstrated the power of canvassing the whole genome with comprehensive
screens of mutant embryos. Even those 鈥渓ethal鈥mutations incompatible
with survival can be characterized by examining the cuticle patterns of
embryos that may or may not have made it through embryogenesis. The cuticle
prep that remained after dissolving away the embryo within retained the body
plan signatures for scoring mutations that affect segmentation and polarity
but would not work for scoring mutations affecting the nervous system. The
hybridoma technology developed by K枚hler and Milstein for monoclonal
antibody generation (K枚hler and Milstein, 1975) offered some hope. Without
any knowledge of the molecules involved in neural development, we could
simply immunize mice with ground up embryos and screen the monoclonal
antibodies based on the staining patterns they yield. That was the plan
anyway; and then there were some accidental findings and lucky breaks in
this venture.
To get started with monoclonal antibody generation, Sandra Barbel, who was
initially planning to join our brand new lab as a technician for a year or
two before returning to her graduate study at UC Davis, learned the
hybridoma technologies with the helpful advice from Lou Reichardt鈥檚 lab.
Sandra has remained and is now our lab manager. Yuh-Nung developed a routine
of listening to his favorite music while going through hundreds of staining
patterns looking for those monoclonal antibodies that appeared to recognize
specific cell types or structures, especially ones in the nervous system.
This way we began the bootstrap approach of using whatever monoclonal
antibodies that emerged from these screens and that could help with the
identification of mutant embryos with abnormal distribution of neurons and
characterizing those mutants molecularly to end up with more markers for
neurons and their precursors that could then be used for additional mutant
screens.
Besides the generation and screening for monoclonal antibodies, serendipity
smiled several times in our haphazard experiments. Early on, when Yuh-Nung
was using horseradish peroxidase (HRP) as a neuronal tracer in the study of
the frog autonomic nervous system, he had a vial of antibody against HRP in
the refrigerator. Because Lily was doing staining of cryostat sections of
fruit flies with antibodies against neuropeptides for no particular reason
beyond a simple curiosity, she reached for the vial of secondary antibody
and ended up with an amazing staining pattern of the entire nervous system鈥
攔ather odd and unexpected given the sparse distribution of peptides in the
vertebrate nervous system. For the next three days, Lily vehemently argued
against Yuh-Nung鈥檚 suggestion that she may have made a mistake somewhere,
until she repeated the experiment using either the secondary antibody meant
for her experiment or the antibody against HRP in the vial on the same shelf
. As Yuh-Nung likes to tell the story, because it was Lily鈥檚 mistake that
led to the surprise finding that antibody against HRP specifically labels
Drosophila and grasshopper neuronal membranes, she was the first author of
the paper on that study (Jan and Jan, 1982a). While we were going through
rounds and rounds of mouse immunization for hybridoma generation, we
immunized some mice with HRP for good measure and recovered a monoclonal
antibody that prominently marked the germ plasm and germ cells and led Bruce
Hay to the molecular characterization of Vasa (Hay et al., 1988). However,
this Vasa antibody does not recognize HRP, and we have no explanation other
than luck for the way we came up with some of the most useful monoclonal
antibodies for our mutant screens.
Even with those neuronal markers, we were still unsure on which part of the
nervous system to focus our attention. Around 1985, we finally realized that
the larval peripheral nervous system (PNS) is a good assay system for
studying neural development. Rolf Bodmer, Alain, Christine, and the two of
us worked out an atlas of the larval PNS (Ghysen et al., 1986; Bodmer and
Jan, 1987). Then, Alain and Christine made a very critical discovery. They
found that mutants of the achaete scute complex (AS-C) displayed a very
striking PNS phenotype: one type of sense organ, the external sensory (es)
organ, was missing but the chordotonal (cho) organs were not affected (
Dambly-Chaudiere and Ghysen, 1987). This was the first, and very nice,
example that a mutation can produce a very clear-cut and neuronal type
specific phenotype, which encouraged us to plunge into serious genetic
screens for mutants affecting PNS development in 1985. That is how genes
like numb and atonal were later discovered.
Shaker Cloning
As we got to know Pat O鈥橣arrell well, we would often stop at the hallway
and chat when we ran into one another. One day Pat was all excited about the
new development with the P-element, the transposon for the mysterious
genetic phenomenon known as hybrid dysgenesis. At the time, Pat was
collaborating with Tom Kornberg on the chromosome walk to clone engrailed
and was raising the possibility of using P-element insertion mutagenesis as
a novel approach to clone Shaker. Pat generously allowed us to do a rotation
in his lab to learn about molecular biology. For the next several years,
there was close collaboration between Diane Papazian, Bruce Tempel, and Tom
Schwarz in our lab and Steve DiNardo and Claude Desplan, who joined Pat鈥檚
lab as postdoctoral fellows, to go after the Shaker gene. At a time when
there were numerous P-elements in a genome prior to the development of
reliable control of movement of these transposons, we settled with the old
reliable chromosome walk for cloning Shaker. As the years for this
chromosome walking, chromosome sitting (we hit an apparently unclonable
region), and chromosome falling off (repetitive sequences doing their trick)
dragged on and other interesting biological questions such as cell cycle
control in the developing Drosophila embryo beckoned, we were left with our
three postdocs Diane, Tom, and Bruce to stick with it to the end.
During the long haul for Shaker cloning and genetic screens for neural
development mutants, it was also payback time after having taken so many
summer courses at Cold Spring Harbor in our graduate student and postdoc
years. We revived the neurobiology of Drosophila course and spent four
consecutive summers at Cold Spring Harbor, starting in 1984, the year we
were expecting the arrival of our son Max. The apartment building for CSH
course instructors was up a gentle slope just about a hundred yards from the
lab, a wonderful arrangement for us to be close to our kids even with the
traditional long hours for the summer courses. Pat joined us in teaching the
course for the first year, and some of our postdocs and students came along
to help out as teaching assistants. Some of the summer course students
decided to do their postdoctoral research with us later on, as in the case
of Ehud (Udi) Isacoff. The scientific nickname Udi can be traced to the
first day when he arrived at Cold Spring Harbor for the summer course and,
while introducing himself to his roommate Claude and struggling with the
French pronunciation of Ehud, he ended up with being stuck with his
childhood nickname Udi.
Howard Hughes Medical Institute and the Birth of Max (1984)
The year 1984 was an eventful one. Scientifically, we were at a low point.
We had been consistently productive ever since our postdoc days (1974鈥982
), but from 1983 to 1986 we had a dry spell. Shaker cloning was very
difficult. Despite three to four years of hard work, we had nothing to show.
Feeling in the dark without knowing what a potassium channel should look
like, we could not even tell whether we were on track to clone the first
potassium channel gene before everything finally clicked in the end. For the
neural development work, we were trying to find our way. We had generated
some very useful markers and developed the embryonic PNS as a promising
system for genetic dissection of neural development, but we had not yet made
any substantial inroad.
During this difficult period, two great events happened to us. One was the
birth on November 7, 1984, of our son Max Huang-Wen Jan. We named him Max in
honor of our PhD advisor Max Delbr眉ck. Following Chinese tradition, all
the kids in Yuh-Nung鈥檚 extended family of Emily and Max鈥檚 generation
share a common given name 鈥淗uang,鈥which means 鈥渂right鈥or 鈥渂
rilliant.鈥For Emily, her specific Chinese given name is 鈥淐hing,鈥
which is an endearing form of 鈥減erson.鈥For Max, his specific Chinese
given name 鈥淲en鈥means 鈥渓iterature鈥or 鈥渃ulture.鈥With Emily and
Max, we are blessed with two wonderful kids, each very talented in a
different way.
Another great thing that happened to us was that somehow we were chosen as
Howard Hughes Medical Institute (HHMI) investigators. At that time HHMI did
not yet have an open competition system, they chose their investigators in a
somewhat mysterious way. In 1984, HHMI decided to start supporting
neuroscience. They picked five institutes, Harvard Medical School, Columbia,
Johns Hopkins Medical School, Salk, and UCSF, and asked these institutions
to nominate potential investigators. UCSF decided to choose neuroscience
faculty who were relatively young and not yet at the rank of full professor.
That meant there was a very small pool of potential candidates. Somehow
UCSF picked us even though we were struggling mightily to get our research
going. HHMI made a huge difference in our research. We are most grateful for
their continued support since July 1, 1984, now more than 29 years and
counting.
Breakthroughs in 1987
Shaker Cloning (1987) to Enable Studies of Potassium Channels One at a Time
Finally, after six years of hard work, the project of Shaker cloning came to
fruition. One day, Diane saw the S4 sequence with the basic residue
arginine or lysine at every third position within an otherwise hydrophobic
sequence that could pass as a transmembrane segment, so she knew we probably
had the real thing. Over the next few months, Diane, Tom, and Bruce
isolated a number of alternatively spliced isoforms of Shaker cDNAs and
their fellow postdoc Les Timpe demonstrated functional expression of voltage
-gated potassium channels in Xenopus oocytes. The Shaker locus turned out to
be quite complex. It is a large gene with several different alternatively
spliced transcripts coding for different proteins that form voltage-gated
potassium channels with a variety of electrophysiological properties. No
wonder it was a beast of a gene for novices like us to tackle. Shortly after
that, we were able to clone its mammalian homologue due to high sequence
homology. It was gratifying that the fly work could be readily extended to
vertebrates. Those results were published in five papers in a nine-month
span (Papazian et al., 1987; Tempel et al., 1987; Schwarz et al., 1988;
Tempel et al., 1988; Timpe et al., 1988).
The remarkable level of evolutionary conservation of potassium Channels
became evident right away. It took us six years to clone the Shaker voltage-
gated potassium (Kv) channels with the chromosome walk and then just a few
months to clone the mammalian homolog of Shaker, Kv1.1. A decade later, the
first case for a channelopathy happened to be episodic ataxia type 1 (EA1)
due to mutations of Kv1.1, as revealed by physicians and biophysicists in
Oregon at the dawning of the decade of the brain (Browne et al., 1994). The
physiological role of Kv1.1 in mammalian motor axons, as demonstrated by
studies of Kv1.1 knockout mice by Tempel鈥檚 lab and Chiu鈥檚 lab, is to
prevent action potentials from bouncing back from nerve terminals (Smart et
al., 1998; Zhou et al., 1998). The recurrent action potential firing after a
single stimulation of mouse motor axons is reminiscent of the recurrent
action potentials in Shaker mutant larval motor axons we showed in our
letter to Katz and later on in two reviews in the Journal of Physiology (Jan
and Jan, 1997, 2012). This hyperexcitability is likely the cause of
myokymia of EA1 patients, the uncontrollable muscle movements of the limb
even after the motor nerve is temporarily isolated via a pressure cuff.
As shown by the studies of Kv1.1 knock-in mutant mice bearing an EA1
mutation by the labs of Jim Maylie and John Adelman, a reduction of Kv1
channel function also causes hyperexcitability of central neurons: Kv1.1
function is important for the cerebellar basket cells to limit action
potential propagation into only a subset of the axonal branches; reducing
Kv1 channel function allows excessive action potential invasion at axon
branch points and compromises the central control of motor activity (Herson
et al., 2003). So, hyperexcitability of neurons in the central nervous
system is likely the basis of the episodic ataxia of EA1 patients.
As the Kv family grew鈥攚ith numerous family members cloned by many labs in
the channel field, we came to realize and appreciate the tremendous
diversity of potassium channels. Looking rather like a quarter of the
poreforming subunit of a sodium channel, Kv channel subunits can form
heteromeric channels with properties distinct from homomeric Kv channels, as
Udi first demonstrated (Isacoff et al., 1990). Moreover, Morgan Sheng and
his fellow postdoc Meei-Ling Tsaur came to the surprise finding that the
potassium channels on the axon and dendrites of a neuron have different
subunit compositions even though they have similar electrophysiological
properties (Sheng et al., 1992). Moreover, the Kv channel expression could
change dynamically with neuronal activity (Tsaur et al., 1992).
Later on, we were amazed to realize that, whereas the evolutionarily
conserved placement of Kv1 channels in the axon is associated with axon
targeting machineries including conserved features of Kv1 channel subunits (
Gu et al., 2003; Gu et al., 2006), the transcripts for Kv1.1 and Kv4.2 are
present in dendrites where synaptic regulation of their local translation
involves molecules linked to tuberous sclerosis and fragile X syndrome,
diseases that greatly increase the risk for epilepsy and autism (Raab-Graham
et al., 2006; Lee et al., 2011). The spatial and temporal variations
combined with the mix-and-match of Kv channel subunits have the potential
for tremendous potassium channel diversity in vivo; studies in our lab and
Bruce Tempel鈥檚 lab provided the first examples of heteromeric Kv channels
in the hippocampus and cerebellum (Sheng et al., 1993; Wang et al., 1993).
The opportunity of back-to-back publications coordinated with our colleagues
is a pleasure we have enjoyed over a score of years, and these exercises
with our lab alumni have been particularly rewarding.
Neurogenesis and Cell Fate Specification: Cut, Numb, and Basic Helix-Loop-
Helix Proteins鈥擠aughterless and Atonal (1987鈥994)
In 1987, around the time we finally succeeded in cloning Shaker, our neural
development work also began to come to fruition. Several of the genes we
started studying during that era provided useful insights into how neuronal
cell fates are specified: cut, numb, daughterless, and atonal.
One of the first neural developmental genes we studied was cut. Rolf Bodmer
and Karen Blochlinger, two postdocs in our lab, discovered that cut
functions as a binary switch between es organ and cho organ fate. Cut is
normally expressed in es organs but not in cho organs. In cut loss of
function mutants, es organs are converted into cho organs (Bodmer et al.,
1987). Conversely, ectopic expression of Cut transforms cho organs into es
organs (Blochlinger et al., 1991). Karen cloned cut and found that it
encodes an unusually large homeodomain containing gene (Blochlinger et al.,
1988). Cut is one of the first examples of homeodomain-containing genes that
when mutated cause cell fate transformation at the single cell level, as
opposed to the homeotic transformation of whole body parts or segments
caused by mutations of the bithorax complex or the antennapedia complex.
Later on, Wes Gruber discovered that cut has another interesting function in
controlling dendrite morphology.
Rolf鈥檚 study of cut led to a very nice by-product. He was trying to
identify additional cut-like genes by sequence homology and identified
another homeodomain-containing gene. It was initially somewhat disappointing
because the gene was expressed in developing mesoderm rather than in the
nervous system and he named it msh-2 for mesoderm specific homeobox
containing gene-2 (Bodmer et al., 1990). Later on in his own lab at the
University of Michigan, Rolf discovered that msh-2 is essential for mesoderm
development in Drosophila, and he renamed the gene tinman because the
mutant lacks heart muscle (Bodmer, 1993). Tinman (the protein encoded by
tinman) turns out to be an evolutionarily conserved protein with important
roles in mesoderm development in Drosophila and in mammals.
Another informative gene is daughterless (da). In da mutants, the entire PNS
is missing. In our lab in 1988, Mike Caudy and Harald Vaessin cloned da and
noticed that there is sequence homology between Da (the protein product of
da), Myelocytomatosis viral oncogene (Myc), and AS-C. Before we wrote up our
paper (Caudy et al., 1988), Lily was at a meeting at MIT and happened to
talk to David Baltimore; she learned that their lab had just cloned a gene
encoding an immunoglobulin kappa chain binding protein E12/E47, which also
has sequence homology with Myc and AS-C. We then sent Baltimore鈥檚 lab the
Da sequence and it turned out that Da is a homolog of E12/E47. They had the
insight to recognize a novel basic helixloop- helix (bHLH) motif, which is a
DNA-binding and protein-dimerization domain, shared by Da, Achaete (Ac),
Scute (Sc), MyoD, and Myc (Murre et al., 1989a). Da normally assembles with
Ac or Sc to form a heterodimer that binds to DNA and regulates the
downstream genes to initiate neural development (Murre et al., 1989b). Da is
ubiquitously expressed, whereas Ac or Sc is expressed in discrete clusters
of cells that prefigure where the future PNS neurons will form. These
findings led Alain and Christine to propose the useful 鈥減roneural gene鈥
concept (Ghysen and Dambly-Chaudiere, 1989; Cubas et al., 1991; Skeath and
Carroll, 1991).
In parallel to the work on Drosophila neurogenesis, there was substantial
progress in understanding vertebrate myogenesis by Harold Weintraub, Eric
Olsen, and others. The way that bHLH factors function as genetic switches to
initiate Drosophila neurogenesis and vertebrate myogenesis turned out to be
remarkably similar (Jan and Jan, 1993; Weintraub, 1993).
Studying the way bHLH factors specify neuronal cell fate led us to approach
an interesting issue: How does neuronal type specificity arise during
neurogenesis? In AS-C mutants, one type of sensory organs, the cho organs,
remain intact. Based on what we knew about how Da and AS-C function to
initiate the development of subtypes of PNS neurons, we postulated that
there is an as yet unidentified gene X, which most likely also encodes a
bHLH protein that can form a heterodimer with Da to initiate cho organ
development. Following this hypothesis, we were able to find this missing
gene and named it atonal, which is indeed required for the development of
cho organs and has all the properties we predicted (Jarman et al., 1993). An
unexpected bonus was our finding that atonal is also essential for
photoreceptor development in the Drosophila compound eye. We found that
atonal is required for the development of the founder photoreceptor R8,
which in turn induces the formation of the other photoreceptors of an
ommatidium of the compound eye (Jarman et al., 1994). This was a
satisfactory finding because it showed that the proneural concept is
generally applicable in Drosophila neural development and provided insights
about the genesis of neuronal cell type specificity.
Atonal is the founding member of the Atonal family of proneural gene
products. Interestingly, the vertebrate homologues are often involved in the
specification of neurons involved in vision and hearing鈥攋ust as in
Drosophila (Kanekar et al., 1997; Bermingham et al., 1999; Kay et al., 2001).
Identifying Founding Members of Another Potassium Channel Family (1993鈥
994) and Searching for Ways Used by Cells to Regulate Channel Number
Although extensive studies of many labs have delineated the extended family
lineage of Kv channels and further linked multiple Kv family members to
human diseases of various tissues including the brain, heart, and muscle, it
also became evident in the early 1990s that certain potassium channels,
such as the inwardly rectifying potassium (Kir) channels important for
controlling heart rate or insulin release, could not be isolated based on
sequence homology to Kv channels. Luckily, that was the time when Yoshihiro
Kubo came to our lab for a two-year stint of postdoctoral research, two
years after he was recruited as a freshly minted PhD from Tokyo University
to join the faculty of the Tokyo Metropolitan Institute.
The circumstances that caused Yoshihiro Kubo to come to our lab in 1991 and
Tadashi Uemura to join our development group as a postdoc a few years
earlier gave us a glimpse of the remarkable dedication of their mentors.
When we and Louis Reichardt became HHMI investigators in 1984 and 1985, HHMI
was having some serious discussions with the Internal Revenue Service (IRS)
about their status as an institute鈥攔ather than a foundation that has to
meet the legal requirement of spending down its endowment by a few percent
each year. So it was important for HHMI to have all our labs moved to one
place to signify the physical presence of an institute at UCSF. Where would
they find the space in the very densely populated UCSF buildings for the
relocation of HHMI labs? The university hospital was among the first
buildings to be built on the UCSF campus in San Francisco in the 1920s,
after the 1906 earthquake. Part of it was leased to HHMI. To reconfigure the
layout of patient rooms to accommodate lab benches, the remodeling entailed
moving the corridor sideways and leaving the pillars in the middle of the
lab. In 1986, our lab moved to this newly renovated space. We spent 18
productive years there鈥攗ntil we moved to the new UCSF Mission Bay campus
in 2004. At our lab-warming party, Mitsuhiro Yanagida from Kyoto University
happened to be visiting UCSF and dropped by to have a drink. Years later鈥攁
fter Tadashi joined our lab, he told us of a highly unusual phone call from
his thesis advisor (Yanagida) urging Tadashi to consider doing his postdoc
in our lab. We must have done something right at that party though we still
do not have the slightest clue what that was. While Tadashi was in our lab,
Yoshihiro Kubo鈥檚 thesis advisor Tomoyuki Takahashi paid a visit to
reminisce about the old times at Harvard's neurobiology department and to
personally endorse the postdoc application of his student Yoshihiro Kubo. As
Tadashi came to the lunchroom, Lily cracked an irreverent joke about the
rivalry between Tokyo University and Kyoto University, Tadashi and Tomoyuki
bowed deeply to each other and exchanged what must have been very formal and
respectful greetings in Japanese. All joking aside, it was our great
fortune that Tadashi and Yoshihiro followed their mentors鈥suggestion to
join our lab because their work opened up two significant new directions for
the study of asymmetric cell division and Kir channels, respectively.
Using the expression cloning approach originally developed by Shigetada
Nakanishi in Japan and our UCSF colleague David Julius while he was a
postdoc with Richard Axel at Columbia, Yoshihiro Kubo cloned the inward
rectifier IRK1 by first injecting pools of cRNA from a cell line to induce
Kir channel expression in Xenopus oocytes and then subdividing the cRNA
pools repeatedly until he found a single cDNA clone for IRK1 (Kubo et al.,
1993a). Using IRK1 cDNA to probe heart and brain cDNA libraries in parallel,
Yoshihiro and his fellow postdoc Eitan Reuveny were looking for G protein-
activated inwardly rectifying potassium channels that mediate
parasympathetic slowing of the heart and slow inhibition in the brain and
ended up with the GIRK1 cDNA from both libraries (Kubo et al., 1993b).
Meanwhile, Nathan Dascal was doing a sabbatical in Henry Lester鈥檚 lab at
Caltech and isolated the same cDNA via expression cloning (Dascal et al.,
1993). Together with ROMK1, a weak inwardly rectifying potassium channel
isolated by expression cloning in Steve Hebert鈥檚 lab (Ho et al., 1993),
these founding members made it possible to identify other Kir family members
including Kir6.2 that corresponds to the pore-forming subunit of the ATP-
sensitive potassium channel that controls insulin release. Whereas the pore-
forming Kv channel subunits have six transmembrane segments, the pore-
forming Kir channel subunits have just two transmembrane segments,
corresponding to S5 and S6 of Kv channel subunits; what is unique with Kv
channels is the first four transmembrane segments that form the voltage
sensor (Kubo et al., 1993a).
Whereas a number of potassium channels contain auxiliary subunits as well as
pore-forming subunits in the Kv or Kir family, the curious arrangement in
the ATP-sensitive potassium channel of four Kir6.2 and four SUR1 subunits in
the transporter family made Noa Zerangue, a truly gifted neuroscience
graduate student, and Blanche Schwappach, the postdoc who was engaged in a
close collaboration with Noa, wonder how a cell could count to eight and
coax these members of two very old protein families to assemble and form
octamers. Having devised a method to detect channel proteins on the cell
membrane independent of channel function, to reveal that only octameric
channels appear on the cell membrane, they found that channels with fewer
than eight subunits are retained in the endoplasmic reticulum (ER) via a
novel ER retention signal in Kir6.2 and SUR1 (Zerangue et al., 1999). The
idea that channel activity could be powerfully controlled at the level of
channel number as well as channel property led their fellow postdoc Zach Ma
to identify other novel motifs for traffic regulation such as ER exit (Ma et
al., 2001).
Numb and Asymmetric Cell Division (1994鈥揚resent)
In our initial screen for genes affecting Drosophila PNS development, one of
the genes that caught our attention was numb because of its interesting
cell fate phenotype. A sensory bristle is derived from a single precursor
that goes through four divisions giving rise to five cells. Each division is
asymmetric. In numb mutants, all the divisions become symmetrical resulting
in a strange sense organ made of four socket cells. When Tadashi Uemura
cloned the gene in our lab, the sequence did not tell us how the gene
product Numb might function (Uemura et al., 1989). After Tadashi returned to
Japan to become a junior faculty member associated with Masatoshi Takeuchi
and to work on cell adhesion molecules, a graduate student (Michelle Rhyu)
continued with the project in our lab and discovered that Numb is
asymmetrically localized to one pole of the sense organ precursor cell in a
crescent shaped cap. Upon cell division, Numb is segregated into one of the
two daughter cells to make them different (Rhyu et al., 1994). Numb
functions, at least in part, by biasing the Notch signaling between the
daughter cells (Rhyu et al., 1994; Frise et al., 1996; Guo et al., 1996).
This process is repeated in all the divisions of the sensory bristle linage.
Michelle also found that Numb is asymmetrically localized in the neuroblast
(NB) divisions.
Numb is the first cell fate determinant identified for asymmetric cell
division in the nervous system. It turns out that Numb is just the tip of
the iceberg. Soon, we and others began to uncover other cell fate
determinants such as Prospero, which is asymmetrically segregated together
with Numb, as well as the complex machinery including adaptor proteins
Miranda and Partner of Numb that function to localize those determinants
asymmetrically during asymmetric cell division (Knoblich et al., 1995; Shen
et al., 1997; Lu et al., 1998). In the late 1990s, our main interest
gradually shifted to dendrite development. Several lab alumni continued to
study asymmetric cell division after they left our lab. (Most notably,
Juergen Knoblich has made many important contributions in his own lab in
Vienna.) Their studies, concerning how Numb and other cell fate determinants
are localized to one of the two daughter cells in order to specify their
fates, have provided useful insights about the mechanisms of asymmetric cell
division. This is still a very active research area.
Dendrite Morphogenesis (1998鈥揚resent)
When one looks at the drawing of neurons by Ramon y Cajal, one cannot help
but be impressed by how beautiful those dendrites are. Different types of
neurons can have strikingly different dendrite morphology. We have always
been fascinated by how different neurons acquire their distinct dendritic
morphology. This research subject is not only scientifically interesting but
also esthetically pleasing. We were waiting for an opportunity to study
this problem. We knew way back in 1987 that a group of Drosophila sensory
neurons known as dendritic arborization (da) neurons have beautiful
dendrites (Bodmer and Jan, 1987). However, at that time, to visualize the
dendrites, Rolf had to dissect the larvae individually and stain them with a
neuronal specific antibody. That was too labor intensive for a genetic
dissection of dendrite development, so we had to wait until there was a
suitable technique. In the early 1990s, Liqun Luo joined our lab. Liqun was
the first postdoc in our lab interested in studying neuronal morphogenesis.
That was the time when people began to appreciate the important role of the
small GTPases Rac/Rho/Cdc42 in regulating actin cytoskeleton in yeast and
fibroblast. Liqun was one of the first to discover the interesting function
of those small GTPases in morphogenetic events in multicellular organisms,
including axon growth, myoblast fusion, and dendritic spine development (Luo
et al., 1994; Luo et al., 1996). Liqun left our lab to start his own lab at
Stanford in 1996 and has done spectacularly well ever since.
In the late 1990s, with the arrival of green fluorescence protein (GFP) and
the Gal4-UAS system, we felt that finally the time was ripe for us to start
studying dendrite morphogenesis. Two new postdocs in our lab, Fen-Biao Gao
and Jay Brenman, decided to take on this project by using a PNS-specific Gal
-4 to drive the expression of GFP in PNS neurons to visualize their
morphology in living larvae. This method worked very well and allowed us to
start a genetic screen for mutants affecting dendrite morphogenesis (Gao et
al., 1999). Although this screen was successful and encouraged us to dive
into dendrite morphogenesis, we were limited by treating all the da neurons
as if they were a homogenous group. As a result, we would pick up mostly
mutations that affected general aspects of dendrite morphogenesis shared by
different neuronal types.
In 2001, a new postdoc, Wes Gruber, joined our lab. For his PhD dissertation
with Jim Truman at the University of Washington, Wes received superb
training in insect neurobiology, which enabled him to recognize that the da
neurons could be divided into four classes based on their dendrite
morphology (Grueber et al., 2002). This greatly enriched our study, allowing
us to gain insights about how different neurons acquire their distinct
dendritic morphology (Jan and Jan, 2010). A particularly nice example is Wes
鈥檚 study of the gene cut that we had studied many years ago. Wes noticed
that Cut expression levels exhibit neuronal type specificity: Class III has
the highest level of Cut, Class IV has a medium level, Class II has a low
level, and Class I has no Cut. By experimentally manipulating the Cut level
in different classes of da neurons, Wes demonstrated that the level of Cut
regulates the extent of dendritic growth in a class-specific manner. Thus,
Cut appears to be a multi-level regulator of class-specific dendrite
morphology, and it can exert its function post-mitotically (Grueber et al.,
2003).
Further, there are common mechanisms used by all four classes of da neurons
to shape their dendritic arbors, for example, repulsion between sister
dendrites鈥攕o the dendrites can spread out, a phenomenon known as 鈥渟elf
avoidance.鈥There are also neuronal type-specific mechanisms. For example,
Class IV, but not Class I, da neuron dendrites exhibit tiling. There are
three Class IV da neurons per hemi-segment, and each neuron鈥檚 dendritic
arbor occupies about one-third of a hemi-segment with very little overlap
with its neighbor. This arrangement is known as tiling because it resembles
tiles covering the floor. Tiling was first discovered in the mammalian
retina, where it is presumably important to ensure complete and non-
redundant coverage of receptive fields. We have continued to study the
mechanisms of tiling (Emoto et al., 2004; Emoto et al., 2006; Han et al.,
2012).
If we label all the dendrites of da neurons, the pattern looks like a
complex entangled mess as one would see in any areas of our brain. One
important lesson we learned from our study is that this complex pattern of
entangled dendrites is actually the superposition of four far simpler
patterns, each made by dendrites of neurons of a particular class. Each sub-
pattern is quite regular. By figuring out how various mechanisms such as
self-avoidance, tiling, and size control help to set up each neuronal type-
specific sub-pattern, we can understand how the complex pattern is generated.
By using da neurons, we have gained some insights over the past 10 years
about dendrite development including how axons and dendrites are made
differently; how a neuron acquires its neuronal type-specific morphology;
how the dendrites of different neurons are organized; how the size of a
dendritic arbor is controlled; how the pruning, remodeling, and regeneration
of dendrites are regulated during development; and how neuronal morphology
affects neuronal function (Jan and Jan, 2010). There is much still to be
learned, and this subject remains the focus of our ongoing research. More
recently, we have also begun to use da neurons to study the molecular
mechanisms underlying mechano-sensation, the least understood among the
senses (Yan et al., 2013).
Characterizing Members of a Novel Channel Family and Ongoing Studies
Although we pursued a range of projects to study potassium channels one at a
time to learn about their physiological functions and their regulation by
neuronal activity, we were aware that even in this post-genome sequencing
era there are still orphan channels with unresolved molecular identity. One
case in point is the calcium-activated chloride channel (CaCC) found in
eukaryotes ranging from green algae to man. Molecular identification of CaCC
was difficult partly because of its broad distribution. To prevent
polyspermia, Xenopus oocytes have robust expression of endogenous CaCC,
which provided the electrophysiological readout for the original expression
cloning of Gq protein-coupled receptors by Nakanishi and Julius. But this
made it impossible to use Xenopus oocytes as the expression system for
expression cloning of CaCC. With the generous help and advice of David
Julius (early on for cloning IRK1 and years later for cloning CaCC), we
searched for an alternative way to attempt expression cloning. Bj枚rn
Schroeder devoted six years of his postdoctoral research to tackling this
problem and resorted to using oocytes of the physiologically polyspermic
Axolotl as the expression system for expression cloning of Xenopus CaCC.
This led to the finding that TMEM16A and TMEM16B of a family of 鈥渢
ransmembrane protein with unknown function鈥encode CaCC (Schroeder et al.,
2008). Using completely different approaches, Oh鈥檚 laboratory in Korea
and Galiatta鈥檚 laboratory in Italy came to the same conclusion that
TMEM16A forms CaCC (Caputo et al., 2008; Yang et al., 2008). Before the
appearance of these three papers for molecular identification of TMEM16A as
CaCC, Jason Rock鈥攚ho was a graduate student with Brian Harfe鈥攔eported
their study of TMEM16A knockout mice. This also took place in the summer of
2008 (Rock et al., 2008), thus hastening the in vivo validation of TMEM16A
as CaCC.
The TMEM16 family, with 10 members in mammals, turns out to be rather
unusual; whereas some family members are anion channels, Huaghe Yang came to
the surprise finding that TMEM16F forms a cation channel. As his fellow
postdoc Andrew Kim worked on generating the TMEM16F knockout mice, we were
surprised to learn that TMEM16F is linked to the Scott syndrome, a bleeding
disorder named after a patient; the loss of function mutation of the TMEM16F
gene is associated with Ms. Scott鈥檚 deficiency in blood coagulation (
Suzuki et al., 2010). In collaboration with our UCSF colleague Shaun
Coughlin, Huanghe and Andrew found that TMEM16F gives rise to a small-
conductance calcium-activated nonselective cation channel in vitro and in
vivo, with a crucial role for calcium-activated lipid scrambling in blood
cells (Yang et al., 2012). There are likely more unexpected features that
will emerge as we revisit the whole range of questions for ion channels鈥攁
bout how ion channels in this new family work and how they modulate neuronal
activity to fulfill their physiological roles.
Our Family and Life Outside of the Lab
Compared to our experience with high school and college entrance exams back
in Taiwan, our kids had a head start with scrutiny of kindergarten
applications that involved observations of the kids and interviews with
their parents, which seemed far more nerve-racking for us than our job
search. As they went through the same schools, Max played on multiple sports
teams while Emily was very much into theater production鈥攕o much so that
she majored in theater at Brown University before getting another bachelor鈥
檚 degree (for arts) at California College of the Arts (CCA). She is working
on getting her Master鈥檚 of Arts from Concordia University in Montreal in
2014. When Max was about to graduate from Princeton University with a major
in molecular biology, he became seriously interested in the physician鈥搒
cientist career path. Because he had none of the pre-med preparations, he
first got his PhD in cancer biology at Stanford and now is on track to get
his MD at UCSF in 2015. It has been just wonderful watching both kids grow
into thoughtful young adults鈥斺passionate about curiosity, creativity,
independence, and the desire to know, understand, and ultimately play some
small part in shaping the human condition鈥濃as Emily put it, about the
way they grew up, in her Artist Interview for the 2011 World of Threads
Festival.
Before our kids left home for college, we rarely went to meetings together (
so there was always one of us at home with our kids). Now that our kids are
grown-ups and we have an empty nest, we have started traveling together for
fun. We have had some wonderful trips in recent years鈥攈iking in the Alps
and on the Milford trail in New Zealand, observing animals in the Serengeti,
and going to the Galapagos with our kids after they finished their college
education. In 2010, we had a wonderful road trip in southern France with our
old collaborators Alain and Christine 36 years after we first met Alain in
Seymour鈥檚 lab. The trip ended with a sublime dinner at Michel Bras, a
Michelin three-star restaurant in the middle of nowhere (see Figure 5). In
2011, we took the opportunity presented by a visiting professorship at the
Chinese Academy of Sciences and did something we had always wanted to do鈥攕
ee Mt. Everest from the base camp in Tibet.
Some Reflections
We have been extremely lucky in our professional lives. One contributing
factor is that, ever since our high school days, we have always been at a
top place with a first-rate peer group: NTU, Caltech, HMS, UCSF, and HHMI.
Peer pressure is one of the most powerful driving forces. Our peers inspire
us. One gets the sense that this is the big league. If you can hold your own
there, then you feel that perhaps you can play in the big league, and you
have a sense of belonging.
We have been extremely lucky to have had fantastic students and postdocs
coming through our lab at UCSF over the past 30 years. To date, more than
135 students and postdocs have come through our lab. Nearly 100 have become
professors or group leaders. Some chose nonacademic careers. Many have
distinguished themselves. We are very proud of their accomplishments. We
learned the philosophy of mentoring from our mentors. When students or
postdocs join the lab, the emphasis should be the nurturing and the
enhancement of their careers, not ours. If they do well, we will benefit as
well because that will attract good people to our lab.
We are also incredibly lucky to have been supported by HHMI for nearly 30
years. HHMI funds people not projects. HHMI investigators do not need to
justify what they plan to do. They are free to change research directions as
they see fit (as we frequently do). On the other hand, we are all held
accountable. There is a rigorous review every five years or so.
Another useful lesson we learned early on from our mentors is the importance
of choosing the problem to study. There is a saying (probably by multiple
people): 鈥淚t takes just as much effort to study something relatively
trivial; you might as well choose some potentially important problems to
work on.鈥Finally, there is a passage from Steve Kuffler鈥檚 last paper (
Kuffler, 1980) that we like very much: 鈥淎fter all, it is ongoing work
which makes one go to the laboratory with a feeling of suspense and cautious
expectation. Although success is rare, we continue in the spirit expressed
by Robert Louis Stevens, that to travel hopefully is better than to arrive.
鈥br />
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