WELCOME TO THE WEDNESDAY
AFTERNOON LECTURE SERIES.
I'M ELLA, [INDISCERNIBLE] A
SENIOR INVESTIGATOR LEADING THE
SECTION ON CELLULAR
COMMUNICATION IN NICHD.
ON BEHALF OF THE WOMEN SCIENTIST
ADVISORS, I AM THRILLED TO
INTRODUCE TODAY'S WALS SPEAKER
DR. JESSICA TREISMAN WHO HAS
REVEALED INCREDIBLE INSIGHTS
INTO THE CELLULAR SIGNALING FROM
STUDIES USING THE DROSOPHILA
MODEL SYSTEM.
DR. TREISMAN IS A PROFESSOR IN
THE DEPARTMENT OF CELL BIOLOGY
AT THE NEW YORK UNIVERSITY GROON
SSMAN SCHOOL OF MEDICINE, SHE'S
A REVERED SCIENTIST IN THE
NEUROAND CELLULAR BIOLOGY
BECAUSE SHE UNCOVERED GENERAL
FUNDAMENTAL MECHANISMS BY WHICH
CELLS COMMUNICATE.
USING THE VISUAL SYSTEM OF THE
FRUIT FLY AS PRIMARY MODEL, HER
GROUP HAS IDENTIFIED NOVEL
COMPONENTS OF SEVERAL SIGNALING
PATHWAYS INCLUDING HEDGE HOG,
WIND, NOTCH, AND EPIDERMAL
GROWTH FACTOR, SHE WORKED OUT
THE ROLE OF THESE NEW COMPONENTS
DURING PATTERNING AND SYNAPSE
FORMATION AND CONNECTED THEIR
MISREGULATION TO HUMAN DISEASES.
HER GROUP ALSO STUDIED HOW
VARIOUS SIGNALS ARE TRANSLATED
INTO CELL DECISIONS FOR
TRANSCRIPTIONAL OR POST
TRANSCRIPTIONAL MODULATION OF
GENE EXPRESSION.
FROM SYNAPTIC PARTNERS SELECTION
TO CONTRACTILE FORCES THAT
CONTROL TISSUE MORPH O GENESIS
TO CELL FATE DETERMINE NATION
AND SYNAPSE ASSEMBLY, HER
INSIGHTS ARE TRULY INSPIRING.
DR. TREISMAN RECEIVED HER
UNDERGRAD DEGREE FROM OXFORD
UNIVERSITY AND HER Ph.D. FROM
ROCKEFELLER UNIVERSITY WORKING
WITH [INDISCERNIBLE].
AFTER A POST DOCTORAL FELLOWSHIP
IN THE LABORATORY OF GENERAL
RUBEIN AT UC BERKELEY, DR. T
REISMAN MOVED BACK TO NEW YORK
IN 1996 TO TAKE UP POSITION AT
FACULTY NYU SCHOOL OF
BIOMOLECULAR MEDICINE.
SHE QUICKLY ROSE THROUGH THE
RANKS AT THE INSTITUTE BECOMING
FULL PROFESSOR THERE IN 2007 AND
DIRECTOR IN TWEBT 20.
IN ADDITION TO HER INSIGHTFUL
RESEARCH, DR. TREISMAN IS A
STELLAR SCIENTIFIC CITIZEN.
SHE SERVED AS NUMEROUS NIH STUDY
SECTION HAS BEEN AN ADHOC MEMBER
OF THE VIEW COMMITTEES, HAS BEEN
A MEMBER OF SEVERAL EDITORIAL
BOARD AND ORGANIZED MANY
IMPORTANT DROSOPHILA
CONFERENCES.
AND I WANT TO LEAVE YOU TODAY
WITH SOMETHING I FOUND
FASCINATING AND INSPIRING.
JESSICA'S MOTHER [INDISCERNIBLE]
WAS A FAMOUS PSYCHOLOGIST WHO
SPECIALIZED IN COGNITIVE
PSYCHOLOGY.
SHE GAVE A WALS TALK IN
JANUARY 1997.
AS FAR AS I AND THE WALS OFFICE
CAN TELL, TODAY MARKS THE FIRST
TIME A DAUGHTER OF A PAST WALS
SPEAKER HAS FOLLOWED IN HER
MOTHER'S FOOT STEPS.
JESSICA THANK YOU SO MUCH FOR
COMING.
WE LOOK FORWARD TO YOUR TALK.
THANK YOU.
[ APPLAUSE ]
>>WELL, THANK YOU VERY MUCH FOR
THE KIND INTRODUCTION AND I AM
ESPECIALLY HONORED TO BE
FOLLOWING IN MY MOTHER'S FOOT
STEPS HERE.
SO TODAY I'M GOING TO TRY TO
TELL YOU ABOUT A QUITE NEW
PROJECT IN MY LAB, IN WHICH
WE'RE TRYING TO UNDERSTAND THE
DEVELOPMENT OF THE DROSOPHILA
CORNEAL LENS AND SEE IF IT CAN
TELL US SOMETHING ABOUT THE
HUMAN CORNEA AND USUALLY I DON'T
HAVE TO SPEND TOO MUCH TIME
CONVINCING A AUDIENCE OF
SCIENTISTS THAT THE CORNEA IS
IMPORTANT BECAUSE I BET THAT IF
MOST OF YOU TOOK OFF YOUR
CORRECTIVE LENSS, YOU WOULD SEE
THIS IMAGE LOOKING SOMETHING
LIKE THIS AND THAT'S BECAUSE THE
CORNEA CONTRIBUTES 70% OF THE
REFRACTIVE POWER OF THE EYE AND
SO DEFECTS IN THE CORNEA, LEAD
TO MANY DIFFERENT KINDS OF
VISUAL REFRACTIVE ERRORS.
SO IF THE CORNEA'S TOO STRONGLY
CURVED, IT FOCUSES THE IMAGE IN
FRONT OF THE RETINA, GIVING RISE
TO MYOPIA, IF IT'S NOT CURVED
ENOUGH, THE IMAGE WILL FOCUS
BEHIND THE RETINA GIVING RISE TO
HYPER OPILOT PROJECTA AND IF
IT'S DISTORTED THEN THIS IS
ASTIGMATISM.
THE FLY CORNEAL LENS ADDED FIRST
GLANCE MIGHT LOOK DIFFERENT FROM
THE HUMAN CORNEA BUT THEY DO
HAVE SOME THINGS IN COMMON AND 1
IS THAT THEY'RE MOSTLY COMPOSED
OF EXTRA CELLULAR MATRIX, SO IF
YOU LOOK AT A SECTION THROUGH
THE HUMAN CORNEA, YOU CAN SEE
THAT ABOUT 90% OF THE THICKNESS
IS THE STROMAL LAYER, WHICH IS
AN EXTRA CELLULAR MATRIX MADE UP
OF COLLAGEN FIBERS AND SMALL
LEUCINE RICH PROTEOGLYCANS WITH
SPORADO SIGHTS AND SEVERAL
LAYERS ABOVE AND BELOW IT AND
THE FLY CORNEAL LENS IS AN
ACELLULAR, EXTRA CELLULAR
MATRIX, IN THIS CASE, THE FIBERS
ARE MADE UP OF KITEN, WHICH IS A
POLYSACCHARIDE AND THEIR
AVAILABLE CUTICLE PROTEINS.
ABOUT YOU WE REALLY KNOW VERY
LITTLE ABOUT THE COMPOSITION OF
THIS CORNEAL LENS IS WHY IT
DEVELOPED SUCH PRECISE
CURVEATURE AND WE BEGAN TO
TACKLE THIS AND 1 OF THE FIRST
STEPS WE TOOK WAS TO TRY TO
UNDERSTAND HOW THE CELLS THAT
SECRETE THIS CORNEAL LENS
DEVELOP ISSUES SO THESE WOULD BE
THE UNDERLYING CO-CELLS AND
PIGMENT CELLS.
SO, THIS IS WHETHER THERE'S A
SIGNIFICANT DIFFERENCE BETWEEN
THE DROSOPHILA AND MAMMALIAN
SYSTEMS.
THE MAMMALIAN DEVELOPED FROM THE
EPITHELIUM AFTER THE LENS IS
INVAGINATED AND IT'S QUITE
SEPARATE FROM THE RETINA WHICH
DEVELOPS FROM THE NEURAL TUBE.
BUT IN FLIES, A SINGLE
EPITHELIUM DISK
THE DISK GIVES RISE FIRST TO
THE 8 PHOTO RECEPTORS IN EACH
UNIT OF THE COMPOUND EYE SO
THESE ARE ALREADY, 2, 5, 3, AND
4, AND 1 AND 6 AND 7.
AND THEN THE SAME EPITHELIUM
WILL GIVE PRIZE TO THE 4 CONE
CELLS AND 2 PRIMARY PIGMENT
CELLS AND A LAT ISOTOPE OF
SECONDARY AND TERTIARY CELLS
WHICH WILL COMPLETE THE CORNEAL
LENS.
AND SO, THIS HAPPENS AS A WAVE
OF DEVELOPMENT PROGRESSES ACROSS
THE IIMAGINAL DISK GIVING RISE
TO CELL RISE AND SUCCESS, AND
THAT MEANS THAT A
UNDIFFERENTIATED PROGENITOR IN
THE INTERIOR OF THIS EYE DISK
CAN GIVE RISE TO A NUMBER OF
DIFFERENT CELL FATES AND THIS IS
A QUESTION MY FORMER STUDENT
CAROLYN MORRISON WAS INTERESTED
IN UNDERSTANDING, HOW IS IT THE
SINGLE PROGENITOR COULD GIVE
RISE TO EITHER PHOTO RECEPTOR
WHICH IS A LIGHT SENSITIVE
NEURON, OR CONE CELL, WHICH IS A
GLIAL CELL THAT CAN SECRETE
CORNEAL LENS COMPONENTS OR A
PIGMENT CELL WHICH WILL PRODUCE
SCREENING PIGMENTS AND RECYCLE
RETINAL INDIVIDUAL CYCLE.
SO CAROLYN HAD 1 CLUE TO START
WITH, WHICH WAS A PROTEIN THAT
WAS DISCOVERED BACK IN 1989 BY
DEVIN MOSES AND THE RUBEIN LAB
AND THIS IS A PROTEIN CALLED
GLASS, IT'S A TRANSCRIPTION
FACTOR AND YOU CAN SEE THAT IN A
GLASS MUTANT THE EYE IS MUCH
SMALLER AND MORE DISORDERED THAN
A WILD-TYPE EYE.
AND IF YOU SECTION THROUGH AN
EYE THAT'S A MOSAIC OF BOTH
WILD-TYPE AND GLASS MUTANT
TISSUE, YOU CAN SEE THAT IN THE
WILD-TYPE REGION, YOU SEE THE
PHOTO RECEPTOR RAB DO MERE SO
THESE BLOCKS ARE THE LIGHT
DETECTING ORGANELLE THAT'S
OBVIOUS TO A WATER CONE OUT OF
SEGMENT BUT IN THE REGION, THESE
RAB DO MERES ARE COMPLETELY
MISSING AND IT'S THOUGHT THAT
THE GLASS RECTIFYS THE CELL
PHOTO MATE.
AND IF YOU LOOK AT THE PHOTO
RECEPTOR GENES THEY SEEM TO BE
COMPLETELY MISSING IN THE
MUTANTS.
SO FOR EXAMPLE, K-ROSETTA AND IN
THE IMAGINAL DISK MISSING IN
GLASS MUTANTS, ROUGH ATOM DOBSIN
1 IN THE ADULT RETINA IS ALSO
COMPLETELY GONE.
SO, THERE'S A COUPLE OF GENES
BUT WE WANT TO GET THEM ALL
COMPLETE PICTURE OF WHAT THIS
WAS COLLECTING AND SO WE
COLLABORATED WITH MARTIN AT
BAYLOR AND KUMA R IN THEIR LAB
AND THEY DID A SINGLE CELL SEQ
ANALYSIS OF WILD-TYPE AND SINGLE
GLASS MUTANT DISKS AND THIS WAS
AT THE END LEVEL STAGE.
SO IN THE WILD-TYPE EYE DISKS
THEY COULD SEE SEPARATE CLUSTERS
REPRESENTING EACH OF THE PHOTO
RECEPTOR CELL TYPES, SO R8, 2
AND 5, 3 AND 4 AND 1 AND 6 AND 7
AS WELL AS THE CURRENT CELLS,
AND EACH OF THESE CLUSTER SYSTEM
ACTUALLY A TRAJECTORY FROM LESS
DIFFERENTIATED ON THE LEFT TO
MORE DIFFERENTIATED ON THE
RIGHT.
IN GLASS MUTANT DISKSOT OTHER
HAND THE ONLY RAJECTORY FOR THE
OTHER WAS THE R8 FOR TRAJECTORY
AND A SMALLER CONE TRAJECTORY.
SO THERE ARE EARLY MARKERS OF
R2, 3, AND 4, FOR THE PHOTO
RECEPTORS BUT NO EXTENDED
TRAJECTORY OF DIFFERENTIATION
AND A 1, 6, AND 7 SEEM TO BE
COLONEL PLETELY MISSING.
--COMPLETELY MISSING.
SO I WILL SHOW A FEW GENES THAT
IS BASED ON.
SO [INDISCERNIBLE] IS A
PARTICULAR GENE, AND WE KNOW
THAT REPRESENTS THE R8 IN
WILD-TYPE AND GLASS MUTANT AND
IF WE LOOK AT EARLY PHOTO
RECEPTOR SPECIFIC GENES SUCH AS
SCRATCH, YOU CAN SEE THAT THIS
IS COMING ON IN ALL THE PHOTO
RECEPTOR TRAJECTORIES AND
WILD-TYPE AND IN SCRATCH, WE SEE
IT IN R8 AND IT ALSO TURNS ON IN
THIS REGION WHERE OUR R23, 5,
AND 4 ARE JUST BEGINNING TO
DIFFERENTIATE.
BUT LATER PHOTO RECEPTOR
SPECIFIC GENES SUCH AS RETAINED
IN THE GLASS MUTANT ARE ONLY
PRESENT IN R8.
SO GLASS SEEMS TO BE NECESSARY
FOR THE DIFREBTIATION OF ALL THE
PHOTO RECEPTORS OTHER THAN R8.
SO THAT'S WHAT GLASS IS
NECESSARY FOR.
CAROLYN WANTED TO SEE WHAT IT
WAS SUFFICIENT TO DO.
AND SO SHE MISS EXPRESSED GLASS
IN A DIFFERENT TISSUE, THE WING
DISK, TO SEE WHAT IT COULD
INDUCE IN THE ECTOPIC SITUATION.
SO HERE THE GREEN CELLS
EXPRESSING BOTH GLASS AND GFP AS
AN IMAGINAL DISK AND SHE SAW
THAT KOPTICAL IMAGESIN, THAT THE
SPECIFIC GENE WAS TURNED ON BY
GLASS.
IN A WILD-TYPE EYE DISK, KOPTIN
WOULD COME ON IF THE CELLS HAD
ALREADY BEEN EXPRESSING NEURONAL
MARKERS SUCH AS ELAV FOR QUITE A
WHILE BUT IN THE WING DISK ELAV
WAS NOT INDUCED BY A TOPIC
GLASS, SO THIS CAN INDUCE THE
PHOTO RECEPTOR GENES BUT IT'S
NOT TRANSFORMING THE CELLS INTO
PHOTO RECEPTORS BECAUSE THEY'RE
NOT TAKING ON A NEURONAL
IDENTITY.
AND THIS ISN'T COMPLETELY
SURPRISING BECAUSE GLASS
EXPRESSION IN THE EYE DISK IS
NOT RESTRICTED TO THE FET O
RECEPTOR NEURONS.
IT'S ACTUALLY EXPRESSED IN ALL
THE CELLS OF THE POSTERIOR
DIFFERENTIATING REGION OF THE
DISK AS YOU CAN SEE HERE.
SO GLASS CAN'T BE SUFFICIENT TO
INDUCE THE PHOTO RECEPTOR NEURON
IDENTITY, THERE MUST BE
SOMETHING ELSE WORKING TOGETHER
WITH IT.
AND WE HAD A GOOD CANDIDATE FOR
THAT WHICH IS THE GROWTH
RECEPTOR SIGNALING PATHWAY WHICH
MATTHEW FREEMAN AND MATTHEW
ROSES AND OTHERS HAD SHOWED
PHOTO RECEPTORS OTHER THAN R8 TO
DIFFERENTIATE.
SO AS THIS DIFFERENTIATION WAVE
MOVES ACROSS THE EYE DISK, TURNS
ON A TONAL WHICH IS PRONEURAL
FACTOR AND THAT SPECIFIES THE R8
PHOTO RECEPTOR AND R8 THEN
PRODUCES SWITS WHICH IS EGF
RECEPTOR AND ITS EGF RECEPTOR
SIGNALING SO IF YOU'RE NOT GOING
TO KNOCK OUT EGFR SIGNALING, YOU
LOSE THE PHOTO RECEPTORS OTHER
THAN R8.
SIMILAR TO THE GLASS MUTANT
PHENOTYPE.
SO THIS IS JUST A SIMPLIFIED
VERSION OF THE EGF RECEPTOR
SIGNALING PATHWAY AND I JUST
WANT TO DRAW YOUR ATTENTION TO
TRANSCRIPTION DEFACTOR DOWN
STREAM OF THE PATHWAY WHICH IS
CALLED POINTED AND AN EARLY STEP
IN THE PATHWAY, AT THE GTP
BINDING REZ WHICH WE CAN USE TO
ACTIVATE THE PATHWAY BECAUSE
THERE'S A ACTIVE FORM OF IT.
SO, WE LOOKEDDA THE WHETHER THIS
PATHWAY COULD SYNERGIZE WITH
GLASS TO INDUCE NEURONAL
DIFFERENTIATION IN THE WING DISK
AGAIN, SO GLASS ALONE DOES NOT
TURN ON THE NEURONAL MARKER
ELAV, IT TURNS ON ELAV IN A FEW
CELLS BUT NOT SIGNIFICANTLY BUT
IF WE EXPRESS BOTH TOGETHER NOW
A LOT OF CELLS TAKE ON THE
IDENTITY AND EXPRESS ELAV.
AND WE CAN SHOW THIS IS
DEPENDENT ON THE TRANSCRIPTION
FACTOR POINTED BECAUSE IF WE
MISS EXPRESS GLASS AND ACTIVATED
REZ IN CELLS THAT ARE MUTANT FOR
POINTED WE LOSE MOST OF THAT
ECTOPIC ELAV EXPRESSION.
SO THERE SEEMS TO BE SYNERGY
BETWEEN IT IS 2 TRANSCRIPTION
FACTORS GLASS AND POINTED.
SO ELAV IS JUST 1 GENE SO MAYBE
I SHOULDN'T MAKE SUCH A BROAD
CONCLUSION BASED ON THAT BUT MY
STUDENT WANTED TO KNOW WHO ELSE
GLASS AND ACTIVATED RAS COULD
INDUCE AND SO SHE DID RNA SEQ ON
THOSE WING DISKS I JUST SHOWED
YOU TO LOOK AT WHAT GENES WHEN
DUCED BY THE SYNERGISTIC
INTERACTION OF GLASS AND RAS,
AND SHE FOUND 256 GENES THAT
WERE SYNERGISTICALLY INDUCED BY
RAS, AND THAT WERE MORE LONE AND
WERE POINTED DEPENDENT AND AT
LEAST 40% OF THOSE GENES HAVE NO
NEURONAL FUNCTIONS SO THIS IS
JUST A FEW EXAMPLES, NUEVERIN 1
AND SCRATCHED AND NEURON
SPECIFIC TRANSCRIPT FACTORS AND
TAG MIN 4 AND DRIVEN, AND
SYNAPTIC VESICLE PROTEINS, A
TRIP IS A CHANNEL, IT'S
IMPORTANT FOR PHOTO
TRANSDUCTION, AND FOR THE
MICROTUBIAL PROTEIN SPECIFIC TO
NEURONS, SO ALL OF THESE ARE
INDUCED MUCH MORE STRONGLY BY
THE COMBINATION OF GLASS AND
ACTIVATED RAS THAN BY OTHER
ALONE.
AND THIS EXPRESSION IS MUCH
REDUCED IN POINTED MUTANT CELLS.
SO NEXT, SUH WANTED TO KNOW HOW
THIS WAS HAPPENING?
IN A MECHANISTIC WAY AND THERE
ARE 2 SORT OF EXTREME
POSSIBILITIES FOR THAT, SO
EITHER THEY COULD BE A DIRECT
SYNERGY, WHICH BOTH GLASS AND
POINTED WOULD BE DIRECTLY
BINDING TO THE PROMOTERS OF ALL
OF THESE DOWN STREAM GENES.
OR ALTERNATIVELY, EITHER GLASS
OR POINTED OR BOTH COULD ACT
PRIMARILY BY INDUCING OTHER
TRANSCRIPTION FACTORS AND THOSE
TRANSCRIPTION FACTORS WOULD THEN
ACT DIRECTLY ON THESE DOWN
STREAM TARGET GENES.
SO TO ADDRESS THIS QUESTION, SHE
USED A TECHNIQUE CALLED TARGETED
DEM ID, THIS IS A WAY TO LOOK AT
TRANSCRIPTION FACTOR BINDING
SETS IN A WAY, SO THE WHYED IS
THAT YOU FUSE THE FACTOR THAT
YOU'RE INTERESTED IN TO THE DAN
E.COLI AND THAT CAN THEN
METHALATE WHERE THE PROTEIN IS
BOUND AND YOU CAN EXPRESS THIS
IN SPECIFIC CELL TYPESSA THE A
VERY LOW LEVEL SO IT DOESN'T
DISRUPT A CELL FATE OR ANYTHING.
AND YOU CAN USE METHYLATION TO
THEN ISOLATE AND SEQUENCE THAT
DNA.
SO SUH DID THIS WITH BOTH GLASS
AND POINTED SO SHE FUSED EACH OF
THOSE TO THE METHYLASE AS WELL
AS EXPRESSING DAM METHYLASE OPEN
CONTROL AND HE DID THIS IN 3
DIFFERENT CELL TYPES, SO
INTERNAL IS EXPRESSED IN THE
FIRST CELLS TO DIFFERENTIATE IN
THE EYE DISK.
LV4S IN THE PHOTO RECEPTORS AND
THE SPARK IN THE CONE CELLS, AND
TO SEE WHAT GENES CAN FIND TO IN
ALL OF THESE DIFFERENT CELL
TYPES.
SO WE JUST TAKE OUT THESE DATA A
COUPLE WEEKS -ING AND SO I CAN'T
REALLY TELL YOU THE FULL
ANALYSIS YET, BUT I JUST WANTED
TO SHOW A FEW PRELIMINARY
CONCLUSIONS SO FIRST WE CAN SAY
THAT GLASS AND POINTED HAVE BOTH
SHARED AND DISTINCT TARGETS SO
FOR INSTANCE BOTH GLASS AND CONE
BIND TO THE LIGAND GENE, AND
THEY CONTAIN AND SHOW NECESSARY
THE SUFFICIENT ENHANCEMENT TO
THE EYE DISK, SO THAT WAS NICE.
SOME GENES LIKE [INDISCERNIBLE]
POINTED BY NOT GLASS.
ALSO IF YOU LOOK AT THE
DIFFERENT CELL TYPES, THERE ARE
SOME GENES THAT ARE REGULATED OR
BOUND BY GLASS IN ALL CELL
TYPES, THE EARLY DIFIENTERATING
CELLS, THE DIFFERENTIATORS AND
THE CONE CELLS, THOSE ARE ONLY
IN THE EARLY CELLS OR ONLY IN
THE NEURONS OR ONLY IN THE CONE
CELLS.
AND SIMILARLY FOR POINTED THERE
ARE SOME GENES THAT ARE BOUND BY
POINTED IN ALL 3 CELL TYPES OR
ONLY IN EARLY CELLS, NEURONS OR
CONE CELLS.
DSM AND TO COME BACK TO THOSE
SYNERGISTICALLY INDUCED GENES,
THERE ARE SOME THAT SEEM TO BE
DIRECTLY REGULATED BY GLASS AND
POINTED BINDING TO THE SAME SITE
SUCH AS [INDISCERNIBLE].
YOU CAN SEE BINDING BY BOTH
GLASS AND POINTED, AND THERE ARE
OTHERS THAT APPEAR TO BE
INDIRECTLY REGULATE SAID BECAUSE
WE DON'T SEE BINDING OF EITHER
GLASS OR POINTED SO THERE MUST
BE A SECONDARY TRANSCRIPTION
FACTOR THAT'S MEDIATING THE
EFFECT OF GLASS AND ACTIVATED
RAS ON EXPRESSION OF NERFIN 1.
SO THESE ARE ALSO EXPRESSED BY
OTHER CELL TYPES IN THE DISKS
AND WE WONDER WHETHER IT MIGHT
HAVE A FUNCTION AS OTHER CELL
TYPES AS WELL AND WE COULD SHOW
THAT IT DOES SEEM TO SO IF WE
LOOK AT THE GENES THAT ARE
INDUCED BY GLASS WHEN THIS TAKES
PLACE IN THE WING DISK, WE CAN
FIND GENES THAT ARE SPECIFIC FOR
CONE CELLS OR PRIMARY PIGMENT
CELLS OR SECONDARY AND TERTIARY
PIGMENT CELLS AND THESE ARE ALL
INDUCED BY GLASS INDEPENDENTLY
FOR RAS ACTIVATION, SO RAS
ACTIVATION DOES NOT FURTHER
INDUCE THEIR EXPRESSION.
AND WE ALSO NEW THAT GLASS HAS
BIOLOGICAL EFFECTS ON THESE
NONNEURONAL CELL TYPES, SO IF WE
KNOCK OUT GLASS AUTONOMOUSLY IN
THE PIGMENT CELLS WE LOSE THE
RED EYE PIGMENT.
AND IF WE MISS EXPRESS GLASSOT
SIGHTS OR BODY, WE CAN SEE
INDUCTION OF RED EYE OR PIGMENT.
SO GLASS SEEMS TO BE SPECIFIC TO
INDUCE SOME ASPECT OF PIGMENT
CELL IDENTITY.
SO AN TONIA [INDISCERNIBLE] A
MASTER STUDENT WANTED TO KNOW IF
GLASS WAS DIRECTLY REGULATING
THESE GENES THAT ARE SPECIFIC
FOR THE NONNEURONAL CELL TYPES
AND SO SHE TRIED TO CLEAR OUT
SOME ENHANCERS UP STREAM EVER
GENES THAT WE KNEW WERE GLASS
DEPENDENT AND HAD PREDICTED
GLASS BINDING SITES.
AND SHE WAS ABLE TO FIND SOME
THAT WERE SPECIFICALLY EXPRESSED
OR SPECIFICALLY DROVE FOR
REPORTER EXPRESSION IN CONE
CELLS, AND OTHERS THAT
SPECIFICALLY DROVE REPORTER
EXPRESSION IN PIGMENT CELLS.
SO HERE WE SEE THE PRIMARY
PIGMENT CELLS IN THE SECONDARY
AND SERBIARY PIGMENT CELLS AND
THIS EXPRESSION IS GLASS
DEPENDENT BECAUSE IF WE MAKE
GLASS MUTANT CLONES AND THESE
RETINAS AND THE GLASS MUTANT
REGIONS ARE MARKED WITH GFP, WE
SEE LOTS OF REPORTER EXPRESSION
DRIVEN BY THESE ENHANCERS.
SO WE STILL NEED TO MUTATE THESE
GLASS SITES TO SHOW DEFINITIVELY
THAT GLASS IS DIRECTLY
REGULATING THESE ENHANCERS BUT
WE THINK THAT THEY WILL BE
USEFUL TO HELP US UNDERSTAND WHY
GLASS WOULD TURN ON DIFFERENT
GENES IN DIFFERENT CELL TYPES.
SO WHY DOES IT TURN DOWN THIS 1
ONLY IN CONE CELLS, THIS 1 ONLY
IN PIGMENT CELLS AND OTHER GENES
ONLY IF PHOTO RECEPTORS.
AND WE MENTIONED THAT THERE ARE
OTHER TRANSCRIPTION FACTORS THAT
BIND TO THESE ENHANCERS AND
ACCOUNT FOR THESE DIFFERENCES.
AND WE DO KNOW A FEW OF THOSE
TRANSCRIPTION FACTORS JUST BASED
ON OUR GUESSES, SO, FOR
INSTANCE, ESCARGO IS A SPECIFIC
CELL TRANSCRIPTION FACTOR, AND
IT SEEMS TO SYNERGIZE WITH GLASS
TO TURN ON 8 HOURS APPROXIMATE
MENTORSHIP SKILL CELL GENES, SO
IF WE MESS EXPRESSION GLASS, IT
CAN INDUCE RED PIGMENTOT
ABDOMEN, BUT IF WE MISS EXPRESS
GLASS IN CELLS THAT ARE MUTANT
FOR ESCAGOT, IT IS NOT ABLE TO
INDUCE THAT MEG MENTORSHIP SKILL
AND ECTOPIC GLASS IN THE WING
INDUCES ONLY A LITTLE BIT OF
PIGMENT BUT IF WE INDUCE IT WITH
GLASS MORE, WE SEE INDUCTION.
SO WE THINK THAT GLASS AND
ESG CELLS MAY GO TOGETHER AND WE
ALSO IDENTIFIED OTHER
TRANSCRIPTION FACTORS THAT MAY
CONTROL PHOTO REPRESENT
SEPTORSOR SPECIFIC OR CONE CELL
SPECIFIC GENES TOGETHER WITH
GLASS.
SO, THAT'S WHERE WE ARE, WITH
THE CELL FATE QUESTION AND NOW I
WOULD LIKE TO TURN TO THE
QUESTION OF HOW THE CORNEAL LENS
ITSELF IS ACTUALLY PRODUCED BY
THESE CELLS.
SO I REALIZE THIS IS QUITE AN
UNDERSTUDIED AREA, WE ACTUALLY
KNOW VERY LITTLE ABOUT THIS
CORNEAL LENS, AND IT IS A
PARTICULAR STRUCTURE SO WE CAN
GET SOME CLUES FROM HOW IT MIGHT
BE COMPOSED BY OTHER CUTICLES
AND THE LARVA CUTICLE, THE BODY
COVERING, AND THAT CUTICLE IS
MADE UP OF 3 CELL LAYERS, SO
THERE'S AN OUTER LAYER ENVELOPE
THAT HAS WAXES AND PROTEINS,
IT'S A PROTECTIVE LAYER AS IN A
PROCUTICLE THAT CONTAINS THE
CHITIN FIBERS AND IN BETWEEN
THERE'S A EPICUTICLE LAYER
MARKED BY PROTEINS AND OTHERS.
NOW IF WE LOOK AT THESE MARKERS
IN THE CORNEAL LENS, CHITEIN,
SEEMS TO BE ON THE LENS, WE
THINK THAT'S WHERE IT MIGHT BE
ON THE CUTICLE TO BE FOUND BUT
WHEN WE MARKED AN ENDOGENOUS
CUTICLE PROTEIN, WE FOUND IT WAS
IN THE CORNEAL LENS AND NOT IN A
CENTRAL LAYER AS WE THOUGHT IT
MIGHT BE.
SO IT'S REALLY NOT CLEAR,
WHETHER THE CORNEAL LENS HAS THE
SAME POSITION AS THE CUTICLE OR
WHAT THE BULK OF THIS LENS IS
ACTUALLY COMPOSED OF.
SO, WITH THAT IT'S HARD TO TELL
HOW IT'S DEVELOPED, THE
PRECISELY CURVED SHAPE AND OUR
FIRST HANDLE ON THIS CAME WHEN
HONG SUH WAS INVESTIGATING
ANOTHER TRANSCRIPTION FACTOR
CALLED BLIMP 1, QUITE RELATED TO
GLASS AND I SHOULD SAY JAIRED
CALL AT HIS UNIVERSITY AND HIS
OPPORTUNITY DEVELOPED SOME OF
THESE RESULTS AT THE SAME TIME
AND WE COLLABORATED WITH THEM.
SO WHEN THEY KNOCKED OUT BLIMP1
BY MAKING THE MUTATION IN CRSPR,
THEY FOUND THAT BLMP 1 MUTANT
CLONES IN THE ADULT EYE HAD A
RATHER SMOOTH AND GLASSY
SURFACE, WHERE YOU COULDN'T
REALLY DISTINGUISH INDIVIDUAL
CORNEAL LENSS AS YOU CAN IN A
WILD-TYPE EYE.
AND IF YOU CUT SECTIONS THROUGH
THIS EYE, YOU CAN SEE THAT
THROUGH THE WILD-TYPE REGIONS,
THE LENS IS CURVED ON BOTH SIDES
SO IT'S BI CONVEX, BUT IN THE
BLIMP 1 MUTANT REGION WHICH IS
MARK INDEED RED HERE, THE
EXTERNAL SURFACE IS COMPLETELY
FLAT, SO THERE'S A CONVEX LENS.
AND ONCE YOU CALCULATED THE
CHANGE IN FOCAL LENGTH THAT THE
SHAPE CHANGE WOULD CAUSE, AND
FOUND THAT THE FOCAL LENGTH IS
VERY SIGNIFICANTLY INCREASED SO
MUCH SO THAT THESE LENSES WOULD
NOT BE ABLE TO FOCUS LIGHT
WITHIN THE WHOLE THICKNESS OF
THE RETINA.
SO A FLY THAT HAD COMPLETELY
BLIMP 1 EYES WOULD NOT BE ABLE
TO SEE WITH MUCH PRECISION.
SO, WE LOOKEDDA THE WHERE BLIMP
1 IS EXPRESSED AND WE FOUND IT'S
MOST HIGHLY EXPRESS INDEED THE
NONNEURONAL CELL TYPES IN THE
RETINA, THE CONE CELLS PRIMARY
PIGMENT CELLS AND THE SECONDARY
AND TERTIARY PIGMENT CELLS AND
IT HAS A PEEK OF EXPRESSION
ABOUT HALFWAY THROUGH THE PUPIL
STAGE. THIS IS THE TIME AT
WHICH THERE'S A BIG PEEK OF
ECDICEIN, IT'S A STEROID HORMONE
THAT CONTROLS DEVELOPMENTAL TIME
NOTHING DROSOPHILA.
AND WE CAN SHOW THAT BLIMP 1 IS
DEPENDENT ON ECDICEIN, SO IF WE
EXPRESS ECDICEIN RNAi TO KNOCK
DOWN THE SIGNALING IN CLONES IN
THE RETINA MARKED WITH GFP, WE
SAW LOSS OF BLIMP 1 EXPRESSION.
OKAY, SO, IT'S A DEVELOPMENTAL
TIMING SIGNAL IN THESE CELLS.
SO WE THOUGHT THAT FLINT 1 WOULD
BE QUIET IN THE CELLS THAT ARE
KNOWN TO SECRETE MAJOR
COMPONENTS OF THE CORNEAL LENS,
SO THE CURRENT CELLS AND PRIMARY
PEG MENTORSHIP SKILL CELLS ARE
DIRECTLY UPDATERNEATH THE CENTER
OF THE LENS AND THEY ARE KNOWN
TO SECRETE CRYSTALLINE AND OTHER
MAJOR CORNEAL LENS COMPONENTS.
BUT ACTUALLY IF WE KNOCK OUT
BLIMP 1 EXPRESSION SPECIFICALLY
IN THOSE CELLS BY EXPRESSING
BLIMP 1 RANAI FOR THAT'S--RNAi
WITH THE CODE IN THE REGION,
THERE IS NO MAJOR CHANGE IN THE
SHAPE OF THE LENS.
SO WE FOUND THAT THE CELLS THAT
ACTUALLY DO REQUIRE BLIMP 1 TO
CONTROL THE CORNEAL LENS SHAPE
WITH THESE SECONDARY AND
TERTIARY PIGMENT CELLS WHICH ARE
FOUND OF THE PERIPHERY OF
CORNEAL LENS, SO IF WE XOK
KNOCKED DOWN 1 SPHSKLY IN THOSE
CELLS, NOW WE DO SEE CORNEAL
LENSES WITH A FLAT EXTERNAL
SURFACE.
AND YOU CAN SEE THIS.
CLEARLY IF WE DO IT IN THE WHOLE
EYE, SO THE OUTER SURFACE OF THE
LENS IS REALLY COMPLETELY FLAT.
SO WE WERE WONDERING HOW IS IT
THAT THESE CELLS THAT RIGHT AT
THE EDGE OF THE LENS COULD HAVE
AN INFERENCE ON ITS SHAPE AND 1
IDEA WE HAD WAS THAT THESE CELLS
THAT ACTUALLY ATTACH TO THE
PERIPHERY OF THE LENS, AND MAYBE
THEY COULD BE EXERTING SOME KIND
OF MECHANICAL FORCE ON THE LENS
THAT WOULD CHANGE ITS SHAPE.
AND IF WE ZOOM IN ON THIS REGION
WHERE THE PIGMENT CELL ATTACHES
TO THE CORNEAL CELL LENSES AND
LOOK AT THE ELECTRONIC
MICROSCOPY, WE CAN SEE THAT
THERE DOES SEEM TO BE A CHANGE
AT WHICH THE POINTS THAT THE
PIGMENT CELLS ARE LATCHED TO THE
CORNEAL LENS SO IT SEEMS TO BE
MORE OF THESE ATTACHMENT SITES
WHEN WE BEING IMOD DOWN 1 IN THE
PIG M-TEBURKEULOSEIS CELLS SO
THAT MAKE ITS PLAUSIBLE THERE
COULD BE CHANGE IN THE FORCES
THAT APPLY TO THE CORNEAL LENS.
BUT IN ORDER TO GET THESE CELLS,
WE NEED TO KNOW WHAT IS
REGULATING THE PEG MINT IN THE
SECONDARY AND PRIME AREAY CELLS
AND SO SUH DID ANOTHER ANALYSIS
IN WHICH BLIMP 1 WAS KNOCKED
DOWN OR OVEREXPRESSED AND SHE
FOUND MANY DIFFERENT TYPES OF
GENES THAT WERE REGULATED BY
BLIMP 1, BUT 1 CLASS OF GENES
THAT CAUGHT OUR EYE WAS A SET OF
GENES THAT [INDISCERNIBLE]
DOMAIN PROTEINS.
SO SOME OF THESE ARE UPREGULATED
WHEN THESE ARE KNOCKED DOWN AND
OTHERS ARE DOWN REGULATED SO
THESE PROTEINS WERE INTERESTING
BECAUSE THEY WERE KNOWN TO
CONTROL THE SHAPE OF OTHER
CUTICLE STRUCTURES, SUCH AS THE
DENTICLES THAT ARE FOUNDOT
VENTRAL SITE OF THE EMBRYO.
SO [INDISCERNIBLE]'S LAB
RETURNED THAT A NUMBER OF
DIFFERENT CP DOMAIN PROTEINS
EXPRESSED IN THESE DENTICLE
PRODUCING CELLS AND EACH 1 HAS A
DIFFERENT SHAPE ON THE EFFECT OF
THE DEPTICLE WHEN YOU MOVE IT.
SO, [INDISCERNIBLE] TARTED
LOOKAL IF THESE PROTEINS
PHENOTYPES AFFECTING THE SHAPE
OF THE CORNEAL LENS, AND THE
MOST STRIKING 1, SHE'S FOUND SO
FAR IS FOR A GENE CALLED DUSKY
LIKE, OR DYL, AND THIS IS A
PROTEIN THAT'S FOUND AT THE VERY
TIP OF THOSE EMBRYONIC DENTICLES
SO EACH PROTEIN IS LOCALIZED TO
A DIFFERENT REGION OF THE APICAL
EXTRA CELLULAR MATRIX.
SO SHE FOUND WHEN SHE MADE DYL
MUTANT CLONES IN THE RETINA
MARKED BY GFP HERE, THE CORNEAL
LENS IS AGAIN CHANGE THE SHAPE
TO THE EXTERNAL SURFACES PLATTER
AND THE INTERNAL SURFACES MORE
DEEPLY CURVED.
AND WE CAN QUANTIFY THIS BY
MEASURING THE OUTER ANGLE BY
MEASURING 2 ADJACENT CORNEAL
LENSES, SO IN BOTH BLIMP 1 AND
DYL MUTANT REGIONS THAT ANKLE
GOES UP TO ALMOST 180-DEGREES SO
ALMOST COMPLETELY FLAT.
AND THE DYL MUTANT PHENOTYPE CAN
BE RESCUED BY REEXPRESSING DYL
IN THE MUTANT CELLS.
AND I DIDN'T SHOW IT HERE BUT HE
ALWAYS MEASURED THE INNER ANGLE
AND SHOWED THAT IT'S DECREASED
IN THE DYL MUTANT TERMS.
SO TO UNDERSTAND HOW THIS CHANGE
AND CHANGE IN SHAPE IS HAPPENING
I WENT BACK TO EARLIER STAGES OF
DEVELOPMENT, SO SHE LOOKED AT
THE MIDPUPIL RETINA, AND AT THIS
STAGE, THESE CELLS FORM A
BEAUTIFUL REPEATING PATTERN AT
THE APICAL SURFACE OF THE
RETINA, SO YOU SEE 4 CONE CELLS
IN THE MIDDLE, SURROUNDED BY 2
PRIMARY PIGMENT CELLS, AND THEN
SECONDARY PIGMENT CELLS ON EACH
EDGE AND TERTIARY PIGMENT
CELLINGS OR BRISTLES AT THE
CORNERS.
SO WHEN SHE MADE DYL MUTANT
CLONES, IT ARE MARKED WITH GFP
HERE, AND DYL PROTEIN IS MARKED
IN RED, YOU CAN SEE THAT DYL
PROTEIN IS LOCALIZED TO THE
APICKA SURFACES OF THESE CELLS,
AND IN THE DYL MUTANT REGIONS,
THOSE APICAL SURFACES ARE ARE
SMALLER.
SO CAN YOU SEE FOR INSTANCE
HERE, THAT THIS MUTANT PRIMARY
PIGMENT CELL IS SMALLER THAN THE
WILD-TYPE 1 ON THE OTHER SIDE OF
THE [INDISCERNIBLE], AND THE
SAME THING HERE, AND IN OTHER
PLACES AND THE COMPLETELY DYL IS
A SMALLER OVERALL.
AND A FEW HOURS LATER, THIS
EFFECT THAT COMES MUCH MORE
PRONOUNCED SO THE DYL MUTANT
GREENE, OR SIGNIFICANTLY SMALLER
THAN WILD-TYPE MIRROR IMAGE
TIDDIA IN THE RETINA AND THEY'RE
NO LONGER IN THE SAME BASAL
PLAIN AND THAT'S BECAUSE APICKA
CONSTRICTION USUALLY GOES LONG
WITH THE CELLS, SOPHISTICATEDY
IF WE CUT ACROSS SECTION AT THE
RETINA AT A SLIGHTLY LATER
STAGE, YOU CAN SEE THE BASAL
SHORTENING OF THE GFP LABELED
MUTANT CELLS MORE CLIERLY SO
THEY'RE BECOMING SHORTER IN THE
APICKA BASAL THAT I MENTIONED IN
THE NEARBY WILE TYPE CELLS, AND
YOU CAN ALSO SEE, AN
ACCUMULATION OF ACTIN, AT THE
APICAL SURFACE SO THIS IS
LABELED WITH MAURICEIN, AN
ACTIVE BINDING PROTEIN AND IT
CAN ALSO SEE THAT THERE'S A
CHANGE IN THE SHAPE OF THE
DEVELOPING CORNEAL LENS WHICH IS
BULGING MORE INWARD THAN THE
WILD-TYPE LENSES WHICH ARE
BULGING OUTWARD.
SO WE THINK THAT THIS SHORTENING
OF THE OMA TIDIUM WILL PULLOT
LENS TO MECHANICALLY CHANGE ITS
SHAPE.
SO OUR MODEL BASED ON THIS IS
THAT DYL AT THE APICAL SURFACE
OF THESE CONE AND PRIMARY
PIGMENT CELLS MAY BE ATTACHING
IT TO THE DEVELOPING CORNEAL
LENS, AND THAT THESE APICAL
SURFACES WOULD BE EXPANDED AND
KIND OF UNDERTENSION, SO THAT IF
YOU GET RID OF DYL, THOSE CELLS
WOULD SHRINK THE APICKA SURFACES
THAT DETACH FROM THE LENS AND
KIND OF CONTRACT DOWNWARDS.
AND WE THINK THAT THE SECONDARY
AND TERTIARY PIGMENT CELLS ARE
STILL ATTACHED TO THE CORNEAL
LENS AND SO THEY WOULD PULL THE
CORNEAL LENS INWARD AS THE
OMIRROR IMAGE TIDDIA CONTRACT.
SO WE WANTED TO TRY AND TEST
THIS MODEL AND TO DO THAT WE
INDUCED APICAL CONSTRICTION IN
AN ARTIFICIAL WAY THAT HAD
NOTHING TO DO WITH DYL SO SHE
MISS EXPRESSED AN ACTIVATED FORM
OF LIGHT CHAIN KINASE IN CLONES
OF CELLS AND MARKED WITH GFP.
AND YOU CAN SEE THE SAME KIND OF
APICAL CONSTRICTION IN NEEZ
CLONES SO THAT THE APICKA
SURFACES OF THE OMATIDDIA THAT
ARE EXPRESSING IN THE MICE IN
LIGHT CHAIN KINASE ARE SMALLER
THAN NEARBY WILD-TYPE OMATIDDIA,
AND WHEN SHE LOOKEDDA THE ADULTS
THAT DEVELOPED, SHE SAW THAT
AGAIN IN THESE MUTANT REGIONS,
THE SHAPE OF THE CORNEAL LENS
WAS CHANGED, SO AGAIN IT WAS
BULGING INWARD MUCH MORE THAN
THE NEARBY WILD-TYPE REGIONS, SO
APICKA CONSTRUCTION IN ITSELF IS
SUFFICIENT TO CHANGE THE SHAPE
OF THE LENS AND INCREASE THE
INNER CURVEATURE.
SO HOWEVER, THERE'S STILL A
PROBLEM WITH OUR MODEL AND THAT
IS THAT DYL IS NOT THERE FOR
VERY LONG, SO IT HAS THIS VERY
SHARP PEAK OF MRNA SUGGESTION
ACCORDING TO FLY BASE AT THE
MIDPUPIL STAGE.
AND WE CAN SEE THE SAME THING,
IF WE LOOK AT THE PROTEIN SO
IT'S HIGHEST LEVEL OF EXPRESSION
IS AT 50 HOURS OF PUPIL
DEVELOPMENT AND JUST A COUPLE
HOURS LATER THERE'S MUCH LESS
PROTEIN AND THEN IT DISAPPEARS
SO DYL ITSELF CAN'T REALLY BE
FORMING A REALLY LONG LASTING
ASHES TACHMENT OF THE CORNEAL
LENS TO THE UNDERLYING CELLS.
SO WE KNOW THAT CP DOMAIN
PROTEINS CAN POLYMERIZE INTO
LONG FILAMENTS, SO THIS IS
SHARING THE STRUCTURE IF YOU'RE
MODULAR PT PROTEIN AND EM
STRUCTURE OF THOSE FILAMENTS
THAT IT FORMS AND WE ALSO KNOW
THAT DIFFERENT CP PROTEINS CAN
FORM FILAMENTS TOGETHER.
AND SO WE THOUGHT THAT PERHAPS
THE DYL MIGHT AFFECT THE
ORGANIZATION OF OTHER DOMAIN
PROTEINS SO WE LOOKED AT A FEW
OF THEM AND I SAY 1, THAT WAS
KNOWN TO FORM FILAMENTS IS
DUMPY, THIS IS A REALLY ENORMOUS
PROTEIN, THAT HAS A CP DOMAIN,
AND [INDISCERNIBLE] SHARING THAT
AT MUSCLE ATTACHMENT SITES IT
FORMS VERY NICE FILAMENTS THAT
CONNECT THE MUSCLE TO THE
TENDON.
SO [INDISCERNIBLE] FOUND THAT IF
SHE LOOKED AT DUMPING IN THE
CORNEAL LENS IT ALSO SEEMED TO
BE FORMING SOME KIND OF
FILAMENTAL STRUCTURE AND THIS
ORGANIZATION WAS DEPENDENT ON
DYL, SO IN DYL MUTANT REGIONS,
WE DON'T SEE THESE FILAMENTS
FORMING.
AND SHE SAW SIMILAR THING WITH 2
OTHER DOMAIN PROTEINS SO
POPOAGAIN FORMS FILMAMENTS IN
WILD-TYPE REGIONS BUT IN THIS
DYL MUTANT REGIONS AND THE SAME
THING FOR TRINITY, WHICH IS
ANOTHER CP DOMAIN PROTEIN, SO
AGAIN THIS SEEMS TO BE FILAMENTS
IN THE WILD-TYPE BUT NOT IN THE
DYL MUTANT REGIONS OF THE
RETINA.
SO THIS MAY BE PART OF THE
ANSWER TO WHAT DYL IS DOING BUT
IT'S--LIKELY MORE COMPLICATED
THAN THAT BECAUSE IF WE ELECTRIC
AT ANOTHER NONCP DOMAIN OPPOSITE
BEHAVIORIAL PHENOTYPENT AS THE
CORNEAL LENS SO THE
[INDISCERNIBLE] LENS THAT IS
FOUND AT THE PERIPHERY OF THE
LENS, THAT DOESN'T SEEM FOCUSED
ON BE A SECRETED OR DEPOSITED
PROPERLY IN MUTANT CLONES SO
IT'S IS STUCK IN THE ZONES.
SO WE THINK THAT DYL IS HELPING
TO FORM A NETWORK OF CPL PROTEIN
FILAMENTS INCLUDING PPOT TRINITY
AND IT MAY ALSO BE FORMING A
CONDUIT FOR THE DEPOSITION OF
OTHER COMOPPOSITE BEHAVIORIAL
PHENOTYPENTS SUCH AS SWEDELE E.
OKAY SO THAT'S ALL WELL AND GOOD
BUT WE HAVEN'T COMPLETELY SOLVED
THE PROBLEM OF THE THIS YET
BECAUSE EVEN THOUGH IT'S
REGULATED BY DYL 1, IT DOESN'T
ACCOUNT FOR THE LENGTH OF 1 ON
THE CORNEAL SHAPE.
BECAUSE IF WE LOOK AT BLIMP 1
CLEARANCE THE SAME STAGE, THE
BLIMP 1 MUTANT CELLS WHICH ARE
MARKED IN RED HERE EXPAND THE
IPICKAL PROFILES SO THAT BIGGER
THAN NEIGHBORING WILD-TYPE CELLS
AND ALSO RATHER THAN JUST
CHANGING, IT DOESN'T SEEM TO
SECRETE MUCH DUMPY AT ALL.
SO BLIMP 1 IS DOING OTHER THINGS
HAN JUST REGULATING DYL.
SO 1 CLASS OF BLIMP 1 TARGET
GENES THAT WE'RE INTERESTED IN
LOOKING AT IS WHETHER THOSE ARE
IMPRESSED BY BLIMP 1.
SO BLIMP 1 IS KNOWN TO ACT AS A
SUPPRESSOR IN SOME OTHER
SYSTEMS.
SO IN BLIMP 1 NUCLEOTIDESITANT
CELLS THESE REPRESSED TARGETS
WOULD BE UPREGULATED AND WE
REASONED THAT IF WE WERE TO DOWN
REGULATE THOSE MESS EXPRESSED
PROTEINS WE MIGHT BE ABLE TO
RESCUE THE FINE O TYPE OF BLIMP
1.
AND YOU KNOW THIS WAS PROBABLY,
TOO SIMPLISTIC BECAUSE THERE'S
SO MANY TARGETS OF BLIMP 1, IT
WAS PERHAPS UNLIKELY WE WOULD
FIND A SIPPING SINGLE 1 THAT
WOULD RESCUE THE PHENOTYPE BUT
WE ACTUALLY DID FIND 1 THAT HAD
A PRETTY STRONG EFFECT.
SO THIS SAY PROTEIN CALLED
CT433.
WE HAVE TO GIVE IT A BETTER NAME
SOON BUT IT WAS UPREGULATED IN
BLIMP 1 KNOCK DOWN RETINAS AND
IT SEEMS TO RESCUE THE PHENOTYPE
SO AGAIN BLIMP 1 CLONES HAVE
FLAT CORNEAL LENSES, BUT IF WE
MAKE BLIMP 1 MUTANT CLONES IN
WHICH WE KNOCK DOWN CT4333, THEY
HAVE MUCH MORE NORMAL CURVEATURE
FROM THE OUTSIDE, THE EYE
DOESN'T HAVE THOSE SMOOTH GLASSY
PATCHES, AND IF WE MEASURE THAT
OUTER LENS ANGLE IT'S RESTORED
TO A MORE NORMAL ANGLE.
AND THIS WAS ESPECIALLY
INTERESTING BECAUSE CT4333, IS
USED TO PROMOTE GTFBI, GIVE RISE
TO MANY HUMAN CORNEAL
DYSTROPHIES.
SO TGFBI AND CT4333 ARE BOTH
CLASSIC DOMAIN PROTEINS AND
WITHIN THOSE CLASSIC DOMAINS
THERE ARE MANY AMINO ACIDS THAT
ARE BOTH CONSERVED BETWEEN THESE
2 PROTEINS AND CAN MUTATE TO
GIVE HUMAN CORNEAL DYSTROPHY.
AND OUR TECHNICIAN IN THE LAB IS
NOW WORKING TO KNOCK IN SOME OF
THESE POINT MUTATIONS THAT GIVE
CORNEAL DYSTROPHY IN THE CT4333
TO SEE IF WE CAN GET AN EYE FINE
O TYPE.
BUT ANOTHER THING WE DO KNOW IS
THAT MANY OF THESE MUTATIONS
THAT GIVE RISE TO CORNEAL
DYSTROPHY HAVE THAT EFFECT
BECAUSE THEY CAUSE ACCUMULATION
OR AGGREGATION OF THE PROTEIN,
SO, THEY CAN EITHER EFFECT ITS
SOLUBLE OR TURNOVER BY UBIQUITIN
MEDIATION OR DISINDUCED
AGGREGATION OF THE PROTEIN AND
CAUSES ACCUMULATION OF THE
CORNEAL DYSTROPHY.
AND SO, WE WANTED TO SEE WHETHER
JUST OVEREXPRESSING CT4333 MIGHT
HAVE AN EFFECT AND IN FACT IT
DOES APPEAR TO FLATTEN THE
EXTERNAL SURFACE OF THE LENS.
SO WE'RE HOPEFUL THAT THERE
MIGHT BE MORE HOMOLOGY BETWEEN
THE CORNEAL LENS OF THE HUMAN
CORNEA THAN MIGHT A--BITS PEESH
AT FIRST GLASS.
AND THAT THINKING ABOUT
CONSERVATION MADE US GO BACK AND
LOOK AT BLIMP 1 BECAUSE THE
[INDISCERNIBLE] LAB IN BLIMP 1
IS EXPRESSED IN THE HUMAN
CORNEA, THIS IS MOUSE CORNEA,
SORRY, BUT IT'S SPECIFICALLY
LOCALIZED TO THE LIMBUS AND THE
THAT'S THE REGION WHERE THE
SURVEATURE OF THE CORNEA
INITIATES, IT'S ALSO WELL ADULT
EPITHELIAL STEM CELLS ARE FOUND.
SO, WE ARE INTERESTED IN
LEARNING MORE ABOUT THE FUNCTION
OF BLIMP 1 IN THE CORNEA, AND SO
WE STARTED TO COLLABORATION WITH
CHECKING AT NYU, AND SHE'S
INTERESTED IN DISEASES OF
CORNEAL CURVEATURE SUCH AS
[INDISCERNIBLE] IN WHICH THE
CORNEA IS TOO CURVED AND SHE
USES BOTH MICE AND HUMAN CORNEAL
ORGANNITES IN ORDER TO STUDY
THIS SO WE THOUGHT THIS WOULD BE
A GREAT SYSTEM IN WHICH WE CAN
LOOK AT THE FUNCTION OF BLIMP 1.
SO WE HAVEN'T GOT VERY FAR WITH
THIS BUT SHE HAS FOUND THAT FROM
HER SINGLE CELL RNA SEQ ANALYSIS
OF BOTH HUMAN CORNEA, AND HUMAN
CORNEAL ORGANOIDS THAT BLIMP 1
IS EXPRESSED IN A SUBSET OF THE
EPITHELIAL CELLS, IN BOTH THE
CORNEAS AND THE ORGANOIDS.
AND HER POST DOC [INDISCERNIBLE]
SHAZ SHATTERED TO LOOK AT BLIMP
1 TARGET GENES BEGINNING WITH A
HUMAN CORNEAL EPITHELIAL CELL
LINE AND THEY'RE STILL WAITING
FOR THE GENOME WIDE ANALYSIS,
BUT HE HAS FOUND AT LEAST 1 GENE
THAT'S REGULATED BY BLIMP 1 IN
BOTH SYSTEMS WHICH IS
[INDISCERNIBLE] AND THAT'S
ENCODED BY HSPT2 IN THE HUMAN
CORNEA AND THAT'S REDUCED WHEN
HE KNOCKS DOWN BLIMP 1 WITH
SiRNA, AND THE GENE IS TROLL,
AND WE ALSO SAW A RIDUKS IN
TROLL WHEN WE KNOCKED DOWN BLIMP
1 IN THE FLY RETINAMENT SO WE
ARE HOPING THERE WILL BE MORE
COMMON TARGETS THAT WILL HELP US
UNDERSTAND WHETHER WE CAN USE
THE CORNEAL LENS TO MODEL THE
HUMAN CORNEA.
SO I HOPE THAT--IT'S STILL EARLY
DAYS BUT WE DO HOPE THAT WE CAN
USE THIS CORNEAL LENS, YOU KNOW
NOT ONLY TO UNDERSTAND HOW WE
CAN MAKE A CURVED STRUCTURE OUT
OF EXTRA CELLULAR MATRIX, BUT
ALSO HOW THE CURVEATURE OF THE
HUMAN CORNEA IS GENERATED AND
WE'RE HOPING TO FIND SOME MORE
COMMON FEATURES, PERHAPS IT WILL
TURN OUT THAT TGF BI IN THE
CORNEA IS REPRESSED BY BLIMP 1.
THERE MIGHT BE RULES FOR
CP-DOMAIN PROTEINS PERHAPS AS CP
IS EXPRESS INDEED THE CORNEAL
ENDOTHELIUM AND IT WILL BE
INTERESTING IF IT TURNED OUT TO
ALSO HAVE A ROLE IN ATTACHMENT
OF THE CORNEA, AND YOU KNOW
WE'RE LOOKING FORWARD TO
INVESTIGATING, POSSIBLY
CONSERVED MECHANISMS IN CORNEAL
DEVELOPMENT AND DISEASE.
SOPHISTICATED WITH THAT I WOULD
LIKE TO THANK MY LAB MEMBERS SO
I TALKED ABOUT THE WORK OF
[LISTING STUDENTS ] AND OUR
COLLABORATORS] LISTING
COLLABORATORS ] AND OF COURSE I
WOULD REALLY LIKE TO THANK NIH
FOR FUNDING THESE PROGECS.
SO THANK YOU.
[ APPLAUSE ]
>>--THAT WAS QUITE A STORY.
I GOT THE FEELING THAT
[INDISCERNIBLE] IS A PROTEIN
THAT MUST BE TIGHTLY REGULATED.
TOO MUCH AND TOO MUCH CAN CREATE
HAVOC IN FIBER FORMATION OF THE
OTHER ZONE [INDISCERNIBLE]
PROTEINS, I WAS WONDERING YOU
SHOWED US HOW THE LOSS OF
FUNCTION CREATE MORE
CONSTRICTION, COULD WE ASSUME
THAT OVEREXPRESSION OF IT WILL
RELAX?
>>SO THAT'S INTERESTING.
THAT IS SOMETHING THAT WE SHOULD
PROBABLY LOOK AT SO WE HAVEN'T
REALLY DONE MUCH ANALYSIS OF DYL
OVEREXPRESSION BUT THAT WOULD
CERTAINLY BE INTERESTING TO LOOK
AT BECAUSE IT DOES SEEM TO BE
EXTREMELY TIGHTLY REGULATED.
SO, YEAH, THANKS.
>>THANK YOU FOR YOUR VERY NICE
AND VERY CLEAR TALK.
THIS IS NOT MY AREA OF EXPERTISE
BUT I WAS WONDING YOU TALKED A
LOT ABOUT HOW THESE PROTEIN
SHAPED THE CORNEA DURING REGULAR
DEVELOPMENT, DO THEY CHANGE IN
THE ADULT AFTER DEVELOPMENT
BECAUSE OBVIOUSLY THERE SEEM TO
BE CHANGES IN THE CORNEAL
STRUCTURE IN HUMANS ANYHOW AFTER
BIRTH AND SO, ARE THIS CHANGES
IN THESE PROTEINS IN THE ADULT
FLY.
>>YEAH, THAT'S ANOTHER THING
THAT WE HAVEN'T REALLY LOOKED AT
MUCH YET, SO, I DON'T THINK
ANYONE HAS MUCH STUDIED HOW THE
CORNEAL LENS MIGHT CHANGE WITH
AGE BECAUSE IT IS JUST THIS
COMPLETELY ACELLULAR, EXTRA
CELLULAR MATRIX STRUCTURE.
AND SO I DON'T KNOW HOW MUCH
TURNOVER THERE IS OF THE
COMPONENTS AND WHETHER THEY KEEP
BEING DEPOSITED IN THE ADULT OR
NOT, THAT IS SOMETHING THAT
WE'VE CONSIDERED LOOKING AT WITH
THIS CT433, 3 MUTATIONS BECAUSE
CORNEAL DYSTROPHY IS SOMETHING
THAT'S DEFINITELY AN AGE RELATED
DISORDER THAT GETS WORSE WITH
AGE AND DIFFERENT AGE HAS
DIFFERENT ONSETS IN HUMANS SO
HERE WE ARE CONSIDERING AGING
THOSE FLIES ONCE WE MAKE THE
MUTATIONS AND SEEING IF AGE HAS
ANY PHENOTYPE.
NTHAT WAS A FANTASTIC TALK.
I HAD A COUPLE QUESTIONS ABOUT
YOUR GLASS BINDING RESULTS.
ONE OF THEM WAS DOES THE LEG
EXPRESS THE CO FACTOR THAT'S IN
THE PIGMENT CELLS FOR THE
PIGMENT SPECIFIC GENES IS THAT
WHY THE TRICK OF THE GLASS WORKS
TO TURN ON THE PIG
M-TEBURKEULOSEIS GENES?
AND THE RPGHT 1 IS I THINK YOU
HAD SEVERAL GLASS TARGETS SHARED
ACROSS ALL CELL TYPES DO YOU
THINK THERE'S A UBIQUITOUSLY
EXPRESSED CO FACTOR ARE FOR
THOSE GENES OR A DIFFERENT IN
THE NATURE OF THE BINDING SITES
THAT LETS GLASS BIND ON ITS OWN
SOMETIMES BUT NEED A CO FACTOR
OTHER TIMES.
>>THOSE ARE GREAT QUESTIONS, SO
ESG IS THE PIGMENT CELL
TRANSCRIPTION FACTOR I MENTIONED
SO THAT IS EXPRESSED IN THE LEG.
IT'S MOSTLY IN THE MOST PROXIMAL
LEG, BUT IT DOES VERY EARLY ON,
IT WOULD BE IN THE LARGER REGION
OF THE LEG DISK AND THAT MAY BE
WHY WE DON'T SEE THIS MEG
MENTORSHIP SKILL NECESSARILY
RESTRICTED TO THE MOST PROXIMAL
PART OF THE LEG, AND YEAH, AS
FOR THE GENES THAT REGULATE THE
GLASS IN ALL CELL TYPES WE
DEFINITELY LOOK MORE CLOSELY AT
WHAT THEY ARE, WE'RE JUST
GETTING THESE DATA RIGHT NOW AND
SEE WHETHER--SO I DON'T KNOW
WHETHER OTHER TRANSCRIPTION
FACTORS WOULD BE NECESSARY TO
BIND GLASS OR WHETHER THEY WOULD
JUST BE NECESSARY FOR
TRANSCRIPTION TO BE ACTIVATED SO
BINDING DOESN'T NECESSARILY MEAN
THAT THERE'S AN EFFECT ON THE
TRANSCRIPTION SO WE'RE GOING TO
HAVE TO REALLY CORRELATE THOSE
WITH OUR DATA ON WHAT GENES
REQUIRE GLASS FOR THEIR
EXPRESSION, YEAH, TO BE ABLE TO
FIGURE THAT OUT.
>>I'VE BEEN ASKED TO REPEAT THE
CME CODE, IT'S 444 PIEF 3.
--44453.
AND DOCTOR IF YOU HAVE ANY
CLOSING REMARKS?
>>WELL IF THERE ARE MOW MORE
QUESTIONS I WOULD LIKE TO THANK
JESSICA ONCE MORE FOR A
FANTASTIC TALK.
[ APPLAUSE ]
AND INVITE ALL OF YOU TO STAY
AROUND A LITTLE BIT FOR MORE
QUESTIONS AND COFFEE AND COOKIES
RIGHT OUTSIDE THE AMPHITHEATRE.
THANK YOU VERY MUCH.