Elucidating the neural circuits in the retina that detectmovement

YONEHARA, Keisuke Professor

Multiscale Sensory Structure Laboratory
Department of Gene Function and Phenomics

2003 Graduated with a degree in Veterinary Medicine, Faculty of Agriculture at the University of Tokyo. 2008 Received his PhD from the Graduate University for
Advanced Studies/National Institute for Basic Biology, then began working as a postdoctoral fellow in the same laboratory. 2009 Postdoctoral fellow at theFriedrich Miescher Institute for Biomedical Research, Switzerland. 2015 Associate Professor and Nordic EMBL Group Leader, DANDRITE Institute, Department of
Biomedicine, Aarhus University, Denmark. October 2021 – present Professor at the National Institute of Genetics

Visual stimuli entering the retina are converted into neural signals and sent to the brain. The retina converts theelements of the color, shape, and motion of visual objects into separate signals. Professor Yonehara has made abreakthrough in this research field by elucidating the mechanism that detects the direction of movement of an object.

Not the start I had imagined

analyzing for other purposes led to an important discovery.

The origin of my research goes back to my graduate school days. It
all started with a discovery in the neural circuits of the mouse retina.
In 2003, after completing my course in veterinary medicine at the
University of Tokyo, I joined Professor Masaharu Noda's laboratory at
the National Institute for Basic Biology (NIBB) as a graduate student.
As a book lover, I read in Professor Susumu Tonegawa’s “Spirit andMatter” (Bunshun Bunko, 1993) that he traveled all over Japan tolook for a graduate school, and I also visited a number of laboratories
and put a lot of energy into choosing a school. The laboratory I
chose, the Noda Laboratory, was perfect for me because I wanted toelucidate neural circuits based on the experimental techniques ofmolecular biology.
I was in high spirits before I began, but unfortunately, I was faced witha lot of hardships one after another. I was studying a group of genesthat work in the neural circuits extending from the retina to the brain,
but I could not obtain the data I wanted. The days went on and on likethat. I even thought of giving up on being a researcher many times.
But then, a stroke of luck came my way. A gene in mice that I was

1experimentally investigate the direction selectivity of retinal cells. It
was impossible to select cells with direction selectivity from the vast
number of cells that make up the retina and record their responses.
However, with the use of GFP-labeled cells, this is now possible.

We were studying the expression pattern of a gene called SPIG1 in
the retina. The gene had been modified with green fluorescentprotein (GFP) so that the resulting protein would fluoresce when
expressed. While looking at a microscopic image of the retina, I
noticed that the fluorescence of the GFP labeling appeared in a
mosaic-like pattern. They were present as spots on the retina, equally
spaced on cells that were far apart from each other. Thinking that this
GFP labeling might capture some important features of the cells in
the retina, I quickly made a decision to change the topic of my
doctoral thesis to the analysis of these cells.

Labeling direction-selective cells
The retina of the eye is a light-sensitive organ. Visual stimuli received
there are transmitted from the retina to the brain. I looked at where
the fluorescent GFP-labeled cells in the retina extended to the brain.
It appeared that they extended to a region called the medial terminal
nucleus (MTN) of the accessory optic system (AOS).
A review of past literature on the MTN of the AOS indicates that it isinvolved in sensing direction selectivity (in the upward and downward
direction) of visual signals.
So what is “direction selectivity?” Before we discuss it, I will explain
the characteristics of the visual circuitry of the retina. It is known thatvisual stimuli received by the retina are extracted into component
signals, such as color, shape, distance, the direction of movement,
and speed of the object being viewed, and sent to the brain. Thismeans that vision selectively detects and perceives information thatis important to the organism, rather than just vaguely perceiving theentire landscape. So, how is it possible to detect that an object in thefield of vision is moving? It has long been known that there are cells
in the retina that selectively respond to motion in a certain direction
and send signals to the brain. Suppose we consider four directions of
motion: up, down, left, and right. It means that there are cells that
respond selectively only when the object moves upward, downward,
to the right, or to the left. This property is called “direction selectivity.”

Quickly drafting a thesis ahead of afast-approaching doctoral defense
In order to prove that my findings were valid, it was necessary to
conduct further experiments using electrophysiological techniques.
However, since electrophysiology was not available in the Noda
laboratory, we applied for a joint research project with Dr. Hiroshi
Ishikane of RIKEN (currently Professor at Senshu University) toinvestigate the actual response of GFP-labeled cells to the directionof motion of visual stimuli in mice.
Meanwhile, I was nearing the end of my doctorate. With the tighttimeline for my degree, I did not have much time to finish thisresearch as a dissertation. So, with a little sadness, I decided topresent my dissertation in two parts. First, without waiting for the
results of the electrophysiological experiments, I summarized theprevious results of the discovery of direction-selective cells in apaper. This was published in PLoS One in 2008 (*1). Later, the results of

I thought that the GFP-labeled cells in the retina might bedirection-selective cells, and to confirm this, I traced the neuralpathway from the brain to the retina, this time in the reverse direction.
To do this, I used a staining technique called retrograde tracing. As aresult, we were able to confirm that the medial nucleus does indeed
lead to ganglion cells in the retina.

the electrophysiology experiments were in line with our expectations
and we were able to demonstrate that GFP-labeled cells are
direction-selective and that they respond only to upward movement.
This paper was published in PLoS One in 2009 (*2).

Here is what was very surprising. Half of the cells traced from thebrain to the retina matched the GFP-labeled cells. However, the otherhalf were not GFP-labeled cells. This means that the circuit to themedial nucleus of the brain extended not only from GFP-labeled cells
but also from non-GFP-labeled cells. So, GFP-labeled cells might
have either upward selectivity or a downward selectivity, and
non-GFP-labeled cells have the opposite selectivity. In other words,
the GFP labeling should identify cells with either one of these types of
direction selectivity. When I realized this, I began trembling at thethought that this would be an important discovery. I still vividly
remember the moment when I looked into the confocal microscope inthe darkroom and jumped up in surprise.

The response to these two papers was very strong, as evidenced by
the response to our poster presentation at a conference of the Society
for Neuroscience. They were also selected as “must-read” papers by
the international paper evaluation system F1000.
I believe the significance of this research result is twofold. One is that
we can now analyze direction selectivity by labeling cells with GFP
using the SPIG1 gene. Since this gene is expressed in mice from theembryo stage onwards, we now have a means of studying how direction
selectivity develops in the retina. F1000 praised this research as “opening
up a new field of genetic analysis of retinal neurons.”

That day became the starting point of my research that continues tothis day. Until that point, no method had been discovered to2direction selectivity in the retina. I did some research on where to
apply as a postdoctoral researcher, and the Botond Roska lab in
Switzerland seemed like the best place to start. The Roska lab was
located at the Friedrich Miescher Institute for Biomedical Research inBasel at the time (he later founded and became director of the Institutefor Molecular and Clinical Ophthalmology in Basel). Dr. Roska wasamong the first to document the optical response of the mammalian
retina using two-photon excitation (TPE) microscopy. The laboratory
had the equipment to conduct TPE microscopy and electrophysiologicalanalyses important for studying neural circuits in the retina, as well as
viral vectors and even optogenetics, which was still a rarity at the time.

The other significant point is that we now know that this upward
direction selectivity is genetically determined in cells. There are many
examples of cells in the nervous system acquiring specific properties
through experience. It had been thought that cells with direction
selectivity could also be of that type, but our study demonstrates that
this is not the case.

Saved by books
I learned the importance of one’s attitude towards research when I
was a postgraduate student at Noda Lab. That was probably themost important thing I acquired during my postgraduate years.
Initially, I tended to stick to my own ideas and did not listen to other
opinions very much. However, in the Noda Lab, I realized that
communication and being open-minded are very important inresearch, and presumably in any job.

I immediately sent an application letter to Dr. Roska with an idea for astudy using GFP-labeled cells. I was invited for an interview and duly
hired. Professor Noda then graciously allowed me to bring my
GFP-labeled mice with me.
On the day I arrived at the Roska lab, I had a six-hour discussion with
Dr. Roska and we decided on several research themes. What is themechanism by which cells acquire the direction selectivity signal?
Previous research had shown that the signals (input signals) thatganglion cells, which make up the retina, receive from surrounding
cells are spatially asymmetric. This asymmetry was hypothesized tobe the mechanism that causes direction selectivity. In a letter to Dr.
Roska, I explained my idea that we could demonstrate the mechanismof direction selectivity acquisition by studying how asymmetric input
signals develop in mice using GFP cells. That became the first research
topic we would undertake. Dr. Roska gave me the idea for the specificexperimental methods to study this. This method used optogenetics,
electrophysiology, and TPE microscopy.

As I said earlier, when I was young, apart from research and sleeping,all I did was reading books. I often sought an answer in books when
distressed or faced with a dilemma. In real life, you are surroundedby only a limited number of people, but you are able to access theexperiences of so many authors through their books. It would be aloss not to learn from the enriching writings of our wise predecessors.
For example, during my postgraduate years, Peter Drucker’s notionthat ‘practice will lead to competence’ had a strong influence on me.
Until I learned this quote, I thought that a person's competence wasdetermined by nature and education, and that the only thing remaining
was to acquire knowledge. But when I encountered the quote, I decided
to change my attitude. Since then, I have tried to determine how I
should act and how I respond by looking at matters more objectively.

I learned these techniques so that I could use them in my own
experiments, and set to work. In the retina, there are cells called“starburst amacrine cells” that send the received visual stimuli toganglion cells. When we examined the input from these cells to
GFP-labeled cells, we found that up to the sixth day of life, signals
are input to visual stimuli from any direction, but in the second weekof life, signals are input only to visual stimuli from a fixed direction(upward). In other words, the study revealed the development of
spatial asymmetry in signals and proved that it confers direction
selectivity on ganglion cells in the retina. The results of the study were
published in Nature in 2011 (*3). I felt that this was the end of the firststage of my research that had begun with papers published in PLoS
One in 2008 and 2009.
Some of the research from my time in the Roska lab was published in
Neuron in 2016 (*4), which many people consider to be the culminationof my research as a whole. The following study identified FRMD7 as
the gene essential for spatial asymmetry.

“Conversations with Goethe”（ Iwanami Bunko, published 1968-69）
left the greatest impression on me when I was a postdoctoral fellow. It
was written about how to be first class - if you would like to create
something new, then you must learn from your predecessors. Thereis nothing new but only in the accumulation of the past. Do not become
self-complacent when seeking originality - these quotes became
guidelines for my actions.

We knew early on that although the SPIG1 gene used for GFP labeling
could identify direction-selective cells, it was unrelated to the cause of
direction selectivity (and spatial asymmetry of the signal). So the race
was on to find out what genes were essential for this mechanism.
I discovered the FRMD7 gene by studying a genetic disease. When
our eyes look out the window on a moving train, for example, they
follow the flowing scenery. This is a natural reflex of the eye moving
to stabilize the visual field, but it is known that this reflexive movement
does not occur in people with a genetic disease called congenital
nystagmus. Seventy percent of people with this genetic disease haveContinuing research in the Roska lab
After completing my PhD, I wanted to further develop my analysis of3

a deficiency in the FRMD7 gene, which causes the disease.
Some of our research results at Aarhus University have not yet beenreleased, but I will talk about a few that have already been published.
Direction-selective cells, the type that send signals to the AOS, tend
to respond strongly when the speed of the object being viewed is slow
and less strongly when the speed is fast. This speed dependence isimportant for controlling the reflex movements of the eye for visual
field stabilization, and we have elucidated its mechanism (*5).

One day, I was looking at a database of experimental results from the
Roska lab. I noticed that the starburst amacrine cells in the retina
expressed this FRMD7 gene. Perhaps congenital nystagmus was adisorder of direction-selective cells in the retina. When I mentioned
this to Dr. Roska, he was excited, and we went directly to a mouse
breeding facility and arranged to create knockout mice lacking afunctional FRMD7 gene. Analysis of these mice revealed that they
lacked direction selectivity to recognize horizontal movement, and wewere able to identify FRMD7 as the cause of this defect.

There is also a type of cell in the retina called the “retinal bipolar cell.”
It was not well understood whether these cells emit direction-selective
signals. When I was in the Roska laboratory, we also conducted
research in this area and concluded that they did not emit such signals,
however, our analysis here has produced experimental results that
overturned our previous conclusion (*6). The response of each individual
synapse was measured with a TPE microscope, and a large amountof data was analyzed using machine learning. We believe that theresults were obtained through more precise analysis. In addition tothis, we also identified the region of the cerebral cortex that signals
from the retina reach by analyzing FRMD7 knockout mice (*7).

The discovery of this gene had another major significance. Previously,
it had been thought that only lower mammals and other vertebrates
had direction selectivity in retinal cells. However, my research provided
the first strong evidence that this was not the case and that it mightalso be present in the human retina. Since then, there has been a lotof work on the analysis of direction selectivity in primate retinas.

Starting up an independent laboratory

Winning a grant in Europe

I was able to write many good papers in the Roska lab and learned alot from Dr. Roska's vision for his research. I thought that the next
step was to finally have my own lab, so I started preparing and sent
my application for a position to about 50 places around the world.

When I started up my own lab at Aarhus University, I obtained research
grants in addition to the start-up funding available from the university.
I will explain about research grants in Europe, including the ones I got
during my post-doctorate.

I received good responses from several laboratories. From among allof the offers, I decided without hesitation to go to Aarhus University in
Denmark. The amount of money available for starting up my laboratory
was outstanding. My research on the retina requires a TPE microscope.
When I went independent, I had to purchase one of these expensive
microscopes, so the amount of start-up money was a top priority forme. I like to plan how I am going to do things, and I choose my actions
based on what is best for me each time. I rarely get lost in anythingother than what I think is most important to me.

There are four types of fellowship that are important when doing
research as a postdoc in Europe: the Human Frontier Science Program
Long-Term Fellowship, the Marie Skłodowska-Curie Individual
Fellowship, the EMBO Long-Term Fellowship, and the Japan Society
for the Promotion of Science Postdoctoral Fellowship for Research
Abroad. Of these, I was selected for the EMBO fellowship and the
JSPS overseas fellowship programs.
For independent researchers, there is also the European ResearchCouncil (ERC), a large research funding agency in Europe. The ERC
has grants for different stages of a researcher's career and is a very
competitive grant. I was fortunate to be selected for the Starting Grantby the ERC. When I started up my lab at Aarhus University, I was able
to use the combined funding from this grant and the Aarhus University
startup budget, which was very helpful.

In 2015, I arrived at the DANDRITE Institute at Aarhus University as
Associate Professor and Nordic EMBL Group Leader. TPE microscopes
use a mechanism that allows fluorescent dyes to be illuminated by
low-energy near-infrared light, so the field of view is not too bright andexperiments can be performed to record how labeled cells respond to
the light. We were able to have this microscope in our own laboratory,
and nothing could have made me happier. With our own microscope,
we were able to modify the system, tweaking the wavelength of thelight stimulus, for example, to make it suitable for us.

4You can apply for an ERC research grant after you have been
selected for a position at a university where you will start your own
company. The review process, including interviews, took about ayear. In writing the application, I first collected three or four
applications from past successful grant recipients to learn how theywrote their applications. Then, I asked a number of people, Nobel
laureates among them, to read and critique my application in order toimprove it. Once you pass the review process, the next step is aninterview. After a 15-minute presentation, there is a 5-minute question
and answer session. Of course, I had been diligently practicing forthe interview.

that mice have a habit of fleeing when they see their natural enemies,
such as hawks and owls, flying overhead. Experiments have confirmedthat when a black circular paper is placed over the mice, they flee tothe shelter. We would like to investigate from the genetic level how this
response is affected by differences in the habitat of each subspecies of
mice, whether there are differences in natural enemies, and whether
there is a relationship with the shape of birds. I have high expectations
that our research will expand from its focus on the retina to higher
level vision systems as a whole.
What I expect from students is …well, they don't have to understandwhat's interesting yet, but I want to recruit candidates who are
motivated to achieve something at a high level and who enjoy
developing themselves. As for postdoctoral fellows, I prefer people
who have a clear mindset of what their goals are.

The time I put the most energy into practicing interviews was when I
was trying to get a position as an independent researcher after the
Roska lab. In European universities, I think most interviews take theform of a symposium where about six candidates are assembled andpresent for about 30 minutes in a symposium format. Every day, I
practiced my presentation over and over again, and afterwards, I
repeated the process of presenting in front of everyone and making
corrections in response to criticism. I also videotaped my presentations
and reviewed them to check my pronunciation and body language. In
addition to Aarhus University, I had interviews at several otheruniversities. The more I practiced, the clearer it was that I was getting
better at what I was doing.

In my youth, I worked hard without breaks since I was well-trained inlaboratories in Japan. Initially, at the Roska Lab, I continued to workoutrageously hard and eventually I became unwell. At that point, I
realized that my health is more important than anything else. Since
then, I try to spend more time with my family and to relax by taking
holidays and going traveling. Looking around the European colleagues
around me, I was so amazed to see that even those with outstandingachievements could all afford to enjoy their research. Therefore, Iemphasize the importance of all my lab members enjoying theirresearch. That is the best way to progress and develop my researchon neural circuits in the retina in the future.

Research expanding to the National
Institute of Genetics
I arrived at the National Institute of Genetics (NIG) in October 2021
and have a dual appointment with Aarhus University until 2023. I wasable to obtain several grants in Japan too, which enabled me topurchase two new TPE microscopes. One of the things we hope to
do at NIG is analyze primates. I would like to start working withmarmosets and see how much of what we found in mice is also
shared with primates, or how different they are. I would also like tolink this to findings that will be useful for understanding human visionand treating diseases.

Interviewer:
Yoshiko Fujikawa, Science writer
Photographer:
Mitsuhiko Kurusu, NIG ORD
August 2022

In addition, I use mice in my research, and the NIG has a large-scale
mouse breeding facility. In addition to the excellent breeding
environment, we have a collection of mouse subspecies from all over
the world. I would like to use these mice to begin research on themechanisms of vision and environmental adaptation. What I mean is5

■ References*1---------■ Expression of SPIG1 reveals
development of a retinal ganglion cell
subtype projecting to the medial terminal
nucleus in the mouse
PLoS One. 2008 Feb 6;3(2):e1533.

*2---------■ Identiﬁcation of retinal ganglion cells
and their projections involved in central
transmission of information about upwardand downward image motion
PLoS One. 2009 Jan 29;4(1):e4320.
*3---------■ Spatially asymmetric reorganization of
inhibition establishes a motion-sensitive
circuitNature. 2011 Jan 20;469(7330):407-410.
*4---------■ Congenital Nystagmus Gene FRMD7 Is
Necessary for Establishing a Neuronal
Circuit Asymmetry for Direction Selectivity
Neuron. 2016 Jan 6;89(1):177-93.
*5---------■ Spatiotemporally Asymmetric Excitation
Supports Mammalian Retinal Motion
Sensitivity
Curr Biol. 2019 Oct 7;29(19):3277-3288.
*6---------■ Direction selectivity in retinal bipolar cellaxon terminals
Neuron. 2021 Sep 15;109(18):2928-2942.e8.

*7---------■ A segregated cortical stream for retinal
direction selectivity
Nat Commun. 2020 Feb 11;11(1):831.

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