OKAY, SO, THANK YOU EVERYONE
WHO WAS BRAVE ENOUGH TO COME
HERE DESPITE THE RAIN AND THANK
YOU TO EVERYONE WHO IS JOINING
US ONLINE.
I GUESS YOU MIGHT BE ABLE TO
HEAR US, WE DEFINITELY CANNOT
HEAR YOU.
AND TODAY IT'S MY PLEASURE TO
INTRODUCE TIM PETROS FROM NIHC--
NICHD, SORRY.
HE IS OUR NLM GROUP SPEAKER AND
MANY OF US DO KNOW TIM AS AN
ACTIVE CONTRIBUTOR TO THE NLM
[INDISCERNIBLE] PROGRAM.
HE HELP US A LOT WITH HIS WORK
AND INSIGHTS IN THE SUMMER AND
FALL AND GOING TO THE BACKGROUND
TIM EARNED HIS Ph.D. IN
COLUMBIA IN 2009.
AFTER THAT HE WAS A POST
DOCTORAL FELLOW AT CORNELL AND
NYU AND IN 2007 -- IS EVERYTHING
OKAY?
-- IN 2007 NIH IN PARTICULAR,
NICHD HAS BEEN VERY FORTUNATE TO
BRING HIM ON BOARD AS A SENIOR
TRACK INVESTIGATOR AND TIM'S
WORK WHICH HAS STARTED I BELIEVE
IS USING MASS
SPCERTAINLY--CERTAINLY C AND HE
HAS EXPANDED INTO THE FIELD OF
GENETIC AND EPIGENERATEDETEC
MECHANISMS THAT REGULATE EARLY
INTERNEURON DECISIONS DURING
NEUROGENESIS AND THE RESULTS OF
TIM'S GROUP'S WORK HAS BEEN PUB
LESHED IN A LOT OF LEADING
JOURNALS IN NEURON, AND SEVERAL
OTHERS.
IN ADDITION TO THAT, TIM
RECEIVED MULTIPLE AWARDS
INCLUDING THE DIVISION OF
INTRAMURAL RESEARCH OF NICHD
SCIENTIFIC DIRECTOR'S AWARD,
MENTOR AWARDS, ET CETERA, ET
CETERA.
THE LIST IS WAY TO ALONG TO
MENTION JUST ENOUGH, THE TITLE
OF TIM'S WALK IS MECHANISMS
REGULATING FETE AND MATURATION
OF FOREBRAIN INHIBITORY
INTERNEURONS AND BEFORE WE JUMP
INTO THAT I WOULD LIKE TO
BRIEFLY TALK TO THOSE OF US WHO
ARE ATTENDING VIRTUALLY -- I
HOPE CAN YOU HEAR ME OR YOU WILL
BE ABLE TO SEE THE PRESENTATION
EMPLOY IF YOU HAVE ANY QUESTIONS
PLEASE DO SEND THEM TO THE
E-MAIL ADDRESS THAT HOPEFULLY
WILL BE PROVIDED TO YOU BY THE
VIDEO CHAT FUNCTION, BUT IF YOU
SEE THE E-MAIL ADDRESS, PLEASE
FEEL FREE TO SEND YOUR QUESTIONS
THERE, IF NOT YOU CAN ALSO USE
THE FEEDBACK BUTTON ON THE
VIDEOCAST PAGE.
THOSE QUESTIONS WILL BE
DELIVERED TO ME SOMEHOW AND I
WILL ASK TIM THE QUESTIONS AT
END OF HIS PRESENTATION.
ALSO, IF YOU ARE INTERESTED IN
JOINING US FOR LUNCH AT NOON,
YOU ARE MORE THAN WELCOME.
WE WILL GO TO THE CAFETERIA IN
NATCHER WHICH IS FORTUNATELY
OPEN TODAY.
WITH THAT, IT IS MY PLEASURE TO
WELCOME TIM TO OUR NLM GROUP
TALK EMPLOY.
>> OKAY, THANK YOU FOR THAT KIND
INTRODUCTION, IT'S NICE TO SEE
SOME FAMILIAR FACES HERE, I
REALLY ENJOYED MY TIME ON THE
TENURED TRACK SEARCH COMMITTEE.
I LEARNED A LOT ABOUT NLM AND
WHAT YOU GUYS DO AS AN
ORGANIZATION AND AGAIN, I AM
VERY HAPPY TO PRESENT MY WORK
HERE.
SO AS WAS SAID, MY LAB FOCUSES
ON MECHANISMS THAT REGULATE FATE
AND MATURATION OF GABBAERGIC
INHIBITORY NEURONS AND THIS IS
WOB OF MY MANY FAILED COVER
IMAGE SUBMISSIONS BUT BASICALLY,
ON THE LEFT, IS A SECTION
THROUGH THE EMBRYONIC MOUSE
BRAIN, ON THE RIGHT IS A SECTION
THROUGH THE ADULT MOUSE BRAIN
AND SO MY LAB TRIES TO
UNDERSTAND HOW YOU GO FROM THIS
SMALL POPULATION OF CELLS IN THE
EMBRYO, TO GENERATE THIS
INCREDIBLY DIVERSE POPULATION OF
GABBAERGIC CELLS THAT POPULATE A
VARIETY OF BRAIN REGIONS NEAR
CRITICAL AND EVERY ASPECT OF
BRAIN FUNCTION.
SO IT GOES WITHOUT SAYING THAT
BRAIN DEVELOPMENT ACCIDENT IN
PARTICULAR BRAIN DEVELOPMENT IS
AN INCREDIBLY COMPLEX AND NEURAL
PROCESS STARTING WITH THE NEURAL
TUBE STRUCTURE AND THEN OVER
MONTHS AND YEARS FORMING THIS
COMPLEX STRUCTURE THAT WE ALL
TRY TO UNDERSTAND AND HOW YOU
GENERATE THIS COMPLEXITY AND
DIVERSITY, I THINK IS 1 OF THE
MOST EXCITING AND CHALLENGING
QUESTIONS IN BIOLOGY.
NOW IF YOU LOOK AT COMPAREISON
OF SPECIES OF DIFFERENT BRAINS,
YOU CAN SAY UP HERE IS THE MOUSE
BRAIN WE USE TO MODEL SYSTEM AND
OVER TIME YOU EVENTUALLY
EVOLUTION AS GIVEN RISE TO HUMAN
BRAIN, WITH OVER 86 BILLION
NEURONS THAT MAKES TRILLIONS AND
TRILLIANS OF SYNAPSES AND THEN
YOU CAN ALSO LOOK AT CROSS
SECTIONS HERE AND AGAIN, HERE'S
THE MOUSE BRAIN, SO EVEN THOUGH
WE CONSIDER IT A MODEL SYSTEM
AND MORE SIMPLISTIC, IT'S
INCREDIBLY COMPLEX IN TERMS OF
CELLULAR DIVERSITY AND
CONNECTIVITY.
BUT THERE'S A LOT WE CAN LEARN
ABOUT IT AND HOPEFULLY APPLY TO
HUMAN BRAIN DEVELOPMENT.
NOW I SAID THE CELLS, I STUDY
THESE INHIBITORY NEURONS, THE
BRAINS CONTAIN EXCITATORY CELLS
AND INHIBITORY CELLS AND THIS
BALANCE IS CRITICAL FOR
MAINTAINING CIRCUIT FUNCTION.
SO THESE CELLS WERE BORN IN THE
VENTRAL PART OF THE BRAIN, THE
BOTTOM PART OF THE BRAIN FROM
THESE GANGLIONIC EVIDENCES AND
HERE IS A 3D VIEW, THE LATERAL,
MEDIAL AND KAWDAL AND MOST OF MY
WALK IS FOCUSED ON THE MEDIAL
AND CAUDALL, AND THESE CELLS ARE
BORN, THEY BECOME POST MITOTIC
IN THESE REGIONS AND THEN THEY
GO AND POPULATE ALL REGIONS OF
THE FOREBRAIN.
WE KNOW A BIT ABOUT THE GENETIC
MECHANISMS, THE GENES THAT DRIVE
INTERNEURONS FROM THESE REGIONS,
THE CG AND MG CELLS THAT ARISE
FROM THESE REGIONS, THEY FORM
DIFFERENT TYPES OF INTERNEURONS
AND THEY'RE MORE OR LESS
OVERLAPPING, SO DEPENDING ON
WHAT MATURE TYPE OF INTERNEURON
YOU ARE, WE KNOW WHETHER YOU
AROSE FROM THE MG OR CG, THESE 2
REGIONS GIVE COMPLETELY DISTINCT
TYPES OF INTERNEURONS.
AND AGAIN AS I MENTIONED THEY
POPULATE THE ENTIRE BRAIN AND
DIFFERENT DENSITIES AND
DIFFERENT MORPHOLOGIES AND
INVOLVED IN PRETTY MUCH EVERY
ASPECT OF BRAIN CIRCUITRY.
>> [INAUDIBLE QUESTION FROM
AUDIENCE ]
>> IN THE PREVIOUS SLIDE?
LET ME GO BACK.
SO THIS WAS ACTUALLY IN UTERY O
OPERATION, SO THIS IS ME
LABELING CELLS HERE WITH A GFP
PLAZ MIDS MID, THAT EXPRESSES
GFP, SO ABOUT 40 DAYS AFTER THE
ELECTROPORRATION, THIS IS WHERE
THE CELLS MIGRATED AND POPULATE,
SO IT'S JUST A REPORTER OF CELLS
THAT WERE BORN IN THIS BRAIN
REGION.
SO IF WE ZOOM IN ON THESE CELLS,
THESE ARE ACTUAL RECONSTRUCTIONS
OF TYPES OF INTERNEURONS AND IT
CAN GIVE YOU A FIELD FOR THE
STRIKING MORPHOLOGIES, SOME
BASKET CELLS HAVE DENSE
EXCELLENT ARBOR ANDS SYNAPSE
LOCALLY AND THERE ARE OTHER CELL
TYPES LIKE THIS MARNATI CELL
WHICH SENDS CELLS AWAY FROM THE
CELL BODY AND FORMS SYNAPSES ON
DISTAL DENDRITES.
IF YOU LOOK AT THIS CARTOON
ISSUES HAVE YOU THIS VERY BORING
PARAMETRAL EXCITATORY CELL IN
GRAY, AND ALL THE COLORED CELLS
WITH THE DIFFERENT TYPES OF
INTERNEURONS THAT CAN SYNAPSE ON
THESE EXCITATORY CELLS SO THIS
GIVES YOU A FLAVOR FOR THE
DIFFERENT MORPHOLOGIES AND
CONNECTIVITY PATTERNS AND AGAIN
HOW DO YOU GENERATE THIS
INCREDIBLE DIVERSITY OF
INHIBITORY CELLS FROM BASICALLY
2 BRAINS REGIONS IN THE
EMBRYONIC MOUSE BRAIN.
NOW IT'S INTERESTING IS THAT
INTERNEURONS MAKE UP ONLY 20-30%
OF THE NEURONS IN THE BRAIN AND
THE EXCITATORY CELLS MAKE UP
70-80%.
IF YOU LOOK AT THE DIFFERENT
SUBTYPES, THIS IS FROM THAL AN
BRAIN INSTITUTE WHERE THEY
CLASSIFIED SUBTYPE SPACE SOLELY
ON TRANSCRIPT ORDER OF MICRONSS,
AND YOU SEE IN THE MOUSE CORTEX,
50% OF THE DIFFERENT CELL TYPES
IN THE BRAIN DEFINED BY GENE
EXPRESSION ARE INTERNEURONS EVEN
THOUGH THEY MAKE UP A SMALLER
PROPORTION OF THE CELLS.
AND THIS ACTUALLY EXPANDS IN THE
HUMAN CORTEX WHERE ABOUT 60% OF
THE CELLS GENETICALLY DEFINED
CELL TYPES ARE INTERNEURONS, SO
THERE'S INCREDIBLY HIGHER
DIVERSITY COMPARED TO THE
MAJORITY POPULATION OF THE
EXCITATORY CELLS.
NOW DYSFUNCTION OF THESE CELLS
IS ASSOCIATED OR IN SOME CASES
CAUSATIVE WITH THE WIDE VARIETY
OF NEURODEVELOPMENTAL DISEASES,
MOST NOTABLY SOMETHING LIKE
EPILEPSY IF THERE'S TOO LITTLE
INHIBITION IN THE BRAIN, IT CAN
BE EXCITABLE AND THAT CAN LEAD
TO EPILEPS EXPE SEIZURE
DISORDERS, BUT IT'S BEEN NOTE
THE NOTED IN A WIDE VARIETY OF
DISEASES AND IN FACT MANY GENES
ASSOCIATE WIDE THESE DISEASES
ARE ENRICHED WITH NEURAL
PROGENITORS SO CELLS AND OFTEN
TIMES IMMATURE NEURONS.
SO THEN WHAT MY LAB HAS BECOME
MORE INTERESTED IN IS THE
GROWING EVIDENCE THAT MANY
PROTURBATIONS AND MUTATIONS, I'M
ACTUALLY AFFECT EPIGENETIC
MACHINE REGULATORY, RATHER THAN
THE GENE CODING SEQUENCE
THEMSELVES.
SO THIS IS KIND OF A DRIVING
THEME OF OUR LAB THAT WE
BELIEVE, WE NEED A BETTER
UNDERSTANDING OF BOTH THE
GENETIC AND GENE REGULATORY
MECHANISMS THAT GUIDE THESE
INITIAL FETE DECISIONS EARLY ON
IN THE EMBRYO AND THAT SHOULD
INCREASE OUR UNDERSTANDING OF
NORMAL DEVELOPMENT AND VARIES
DISEASE ETIOLOGIES.
SO WHEN THERE ARE PROTURBATIONS
IN THESE GENES WHAT GOES ON
EARLY ON THAT EVENTUALLY
MANIFEST INTO A DISEASE
PHONOTYPE?
AS I WILL TELL YOU ABOUT A
COUPLE STORIES TODAY.
THE FIRST IS WE WANT TO DEFINE
WHAT WE CALL AN EPIGENOME ATLAS,
THAT SETS THE GROUND TRUTH FOR
HOW THESE CELLS LOOK FROM THESE
DIFFERENT BRAIN REGIONS IN THE
EMBRYONIC BRAIN AND ONCE WE HAVE
THE BASE LINE, WE'RE ABLE TO SEE
HOW PROTURBATIONS AFFECT THIS
DEVELOPMENT.
SO I WILL TELL YOU ABOUT A
VARIETY OF TECHNIQUES WE USE TO
BASICALLY KIND OF DEFINE THE
GROUND TRUTH OF THESE CELLS,
MANY CELL SEQUENCING ASSAYS AS
WELL AS BULK SEQUENCING ASSAYS
AND ONCE WE ESTABLISHED THIS,
THIS,A LOWSITOUS PERTURB THE
SYSTEM WITH GENETIC MUTATIONS TO
UNDERSTAND HOW ALTERATIONS OF
EITHER GENE REPRESSION OR GENE
ACTIVATION, CAN AFFECT CELL
FATE, CELL FUNCTION AND
ULTIMATELY ANIMAL BEHAVIOR AND
LASTLY IF I HAVE TIME, I WILL
TELL YOU ABOUT NEW DATA LOOKING
AT ONCE TRANSCRIPTION FACTOR
THAT'S HIGHLY CORRELATED WITH
NEURODEVELOPMENTAL DISEASE AND
WHAT INSIGHTS WE HAVE FROM OUR
MOST RECENT STUDIES.
SO A LOT OF OUR EXPERIMENTAL
WORK FLOW FOLLOWS THIS PARADIGM.
WE HAVE OUR EMBRYONIC MOUSE, IN
A PARTICULAR AGE WE'RE
INTERESTED IN, WE CAN THEN OPEN
UP THE BRAIN, HEMISECTIONAL
ANALYSISSED AND LOOK INSIDE.
THIS IS OBVIOUSLY STAINED AND
COLORED BUT YOU CAN ACTUALLY SEE
THE STRUCTURES JUST BY EYE
WITHOUT THE STAINING, THERE ARE
BUMPS INTO THE VENTRICULAR SPACE
SO IT'S VERY EASY TO ACCESS
THESE AND DISSECT OUT THE MG OR
THE CGE AND HERE IT IS IN
CARTOON FORM.
SO WE CAN DISSECT OUT THESE
REGIONS, PREPARE SINGLE CELL OR
SINGLE NUCLEI SUSPENSIONS, OFTEN
FACT SORT THEM, PUT THEM THROUGH
OUR 10 X SYSTEM TO DO SINGLE
CELL SEQUENCING OR ALTERNATIVELY
DO SOME BULK SEQUENCING WITH
OTHER ASSAYS AND THEN USE NICHD
SEQUENCING CORE AND THEN WHEN
MOY LAB SPENDS MOST OF THEIR
TIME DOING IS BIOINFORMATTICS
FIGURING OUT TO YOU MASSAGE THE
DATA AND MANIPULATE IT TO TRY TO
UNDERSTAND BIOLOGY FROM THESE
COMPLEX DATA SETS AND THIS IS A
FAIRLY ACCURATE REPRESENTATION
OF WHAT MY POST DOCS THINK ABOUT
DAY-TO-DAY AS THEY WORK ON THIS
DATA SET.
SO, THERE HAVE BEEN A LOT OF
WORK THAT HAD LOOKED AT
TRANSCRIPT
TRANSCRIPTOMES FROM THESE BRAIN
REGIONS BUT WE WANTED TO LOOK 1
STEP HIGHER AT CHROMATIN
ACCESSIBLE AND BED HOW THE
CHROMATIN LOOKS IN THESE BRAIN
REGIONS THAT GIVE RISE TO
DISTINCT CELL TYPES.
SO COUPLE YEARS AFTER I JOINED
HERE, 10 X DEVELOPED THE
COMMERCIALLY AVAILABLE SINGLE
CELL, ATAQ SEQ PLATFORM, YOU
COULD DO BULK AATTACK SEQ, BUT
IN HETEROGENEOUS ENVIRONMENT
SUCH AS THE DEVELOPING BRAIN
IT'S NOT THAT INFORMATIVE.
SO WHAT ATTACK SEQ IS, THE USE
THE SEQUENCING THAT IDENTIFY
OPEN REGIONS OF CHROMATIN, IT
HAS TAGS, IT CAN CUT THIS DNA SO
THAT YOU'RE LEFT WITH THE PIECES
OF DNA THAT REPRESENT REGIONS OF
CHROMATIN AND THEN YOU CAN
SEQUENCE IT WITH THESE TAGS, SO
WE PERFORM ATAC SINGLE CELL
SEQUENCING ON THESE BRAIN
REGIONS SO WE SECTIONED OUT THE
LG, CG, AND MG, AND THIS GIVES
WAY TO OVERLAPPING NEURONS.
WE PREP THE SAMPLES AND THEN
BASICALLY YOU HAVE THE READ OUT
WHERE YOU HAVE SEQUENCES,
SEQUENCE READS THROUGHOUT THE
GENOME AND WHAT YOU'RE LOOKING
FOR IS LEGIONS WHERE THERE'S A
HIGH,AMOUNT OF SEQUENCE READS IN
A VARIETY OF CELLS AND THEN THAT
WOULD BASICALLY BE ABOVE THE
THRESHOLD YOU WOULD CALL A PEAK
AND THIS IS A REGION THAT IS
ACCESSIBLE IN THE SINGLE CELL OR
THE GROUP OF CELLS THAT WE'RE
LOOKING AT.
-- EACH DOT YOU'RE LOOKING AT
REP REISN'TS CHROMATIN
ACCESSIBLE, RATHER THAN GENE
EXPRESSION AND YOU CAN SEE THAT
THE CELLS FROM EACH REGION KIND
OF CLUSTER TODAY, YOU HAVE
EXCITATORY CELLS IN THE CORTEX,
MG CELLS OVER HERE AND THEN LG
AND CG FOR WHICH WE KNOW LESS,
THERE'S LESS DISTINCT
DIFFERENCES IN GENE EXPRESSION
PROFILES, THEY ACTUALLY KIND OF
CLUSTERED TOGETHER HERE AND YOU
CAN PERFORM DIFFERENT TYPES OF
ANALYSIS JUST LIKE YOU COULD
WITH SINGLE CELL RNA SEQ, AND
YOU CAN HAVE REPEAT CLUSTERS.
IN THIS CASE WE KNOW HOW THESE
CELL DIVISIONS WORK AND WE CAN
KIND OF DEFINE DIFFERENT
PROGENITORS IN HERE AND THEN YOU
CAN ALSO DID PSEUDOTIME TO LOOK
AT THE DEVELOPMENT OF THESE
REGIONS IN THESE CELLS ACROSS
NEUROGENESIS.
NOW IN THIS CASE, WHAT WE'RE
LOOKING AT HERE IS HEAT MAPS
SHOWING REGIONS OF PROMOTERS, OF
VARIOUS GENES THAT ARE
ACCESSIBLE, SO NESTIN IS A GENE
THAT'S EXPRESSED IN RADIAL GLIAL
CELLS, VERY IMMATURE CELLS AND
YOU CAN SEE THAT THE ACCESSIBLE
OF THE NESTIN PROMOTER IS
RESTRICTED TO THESE CELLS THAT
ARE THE RADIAL GLIAL CELLS AND
AS THE CELLS GO FURTHER ALONG TO
DEVELOPMENT, THIS LOCI GETS SHUT
OFF AND BECOMES CLOSED SO CAN
YOU DO THIS WITH VARIOUS GENES
OVER TIME AND YOU CAN LOOK AT
VARIOUS GENES THAT HAVE SPATIAL
EXPRESSION, SO NKX2.1, I WILL
COME BACK TO YOU, THIS IS THE
SPECIFIC GENE, CAN YOU SEE THE
PROMOTER IS ONLY ACCESS CLINICAL
COMMUNITY IN THE MGE AND IT'S
CLOSED OFF IN THE OTHER BRAIN
REGIONS.
AND THEN THERE ARE OTHER WAYS TO
LOOK AT THIS, IF YOU LOOK AT THE
PUTATIVE CLUSTERS AT WHICH THERE
IS ABOUT 25, YOU CAN LOOK AT
CHROMATIN ACCESSIBLE AND
CLUSTERS HERE BASED ON SPATIAL
LOCATION, SO CAN YOU SEE AGAIN
THE NK 2.1 PROMOTER IS MAINLY
ACCESSIBLE IN THE MGE AND THE
ABSENCE OF OTHER CLUSTERS AND
AGAIN, YOU CAN LOOK AT THAT IN
BOTH SPATIAL DIVISIONS AND
DEVELOPMENTAL TIME ACCESS HERE.
NOW THIS IS KIND OF A FAIRLY
COMPLEX IMAGE BUT IT'S PRETTY
GOOD SUMMARY OF HOW THE
DIFREPRESENTIALLY ACCESSIBLE
PEAKS LOOK AT OUR DATA SET, SO
IN THIS CASE, THE TISSUE IS
BROKEN DOWN BY THESE COLOR BARS
UP HERE, THESE ARE DIFFERENT
STAGES OF NEUROGENESIS HERE, AND
WHAT WE DID IS BASICALLY TOOK
THE 25 OR SO CLUSTERS WE HAD,
PUTATIVE CELL CLUSTERS AND DID
UNBIASED HIERARCHICAL CLUSTERING
SO WE CAN IDENTIFY CLUSTERS THAT
WERE MOST CLOSELY ASSOCIATED
WITH EACH OTHER AND SO WHAT YOU
CAN SEE HERE, IF YOU LOOK IN THE
UPPER LEFT, THERE'S 4 CLUSTERS
HERE, THESE ARE ALL CORTICALE
APICAL PROGENITORS SO THEY'RE
ALL FAIRLY SIMILAR AND YOU CAN
SEE THERE'S ACTUALLY FAIR --
SOME SIMILARITY BETWEEN THE
DIFFERENTIALLY ACCESSIBLE PEAKS
IN THESE DEFINITE CLUSTERS AND
THAT MAKES SENSE BECAUSE THEY'RE
ALL VERY SIMILAR CELL TYPES,
CONVERSELY IF YOU LOOK AT
DIFFERENCES BETWEEN DIFFERENT
BRAIN REGIONS AND DIFFERENT CELL
TYPES, YOU SEE THERE'S A LOT
LESS SIMILARITY HERE SO WHAT WE
SEE IS THAT THE ACCESSIBILITY
PROFILES OF THESE CELLS ACTUALLY
MATCHES PRETTY WELL WITH BOTH
GENE EXPRESSION AND THERE'S
QUITE DISTINCT DIFFERENCES
BETWEEN BOTH RAIN BREEJIONS AND
BETWEEN DIFFERENT CELL TYPES AND
DEVELOPMENT.
SO TO TAKE 1 BRIEF TANGENT, AS
WE'RE DOING THE DATA WE IN THE
LAB GENERATE RNASEQ DAILY BASIS
THEA OF THE 4 BRAIN REGIONS AND
WE DID THIS BECAUSE WE WANTED TO
HAVE BOTH TRANSCRIPTIONAL DATA
AND CHROMATIN ACCESSIBLE IN
THESE BRAIN REGIONS AND SO, I
WON'T GO THROUGH ALL THIS BUT WE
FOUND DISTINCT EXPRESSION
PROFILES IN THESE CELLS BETWEEN
BRAIN REGIONS AND HERE'S
EXAMPLES FOR LG, MG, AND CG, AND
THERE'S ALSO INITANCES WITHIN
THE SINGLE BRAIN REGION, AND YOU
SEE 1 THAT'S ENRICHED IN THE
DORSAL, AND YOU SEE 1 THAT'S
ENRICHED IN THE VENTRAL KRRK GE
AND WE HOPE AND WE THINK THAT
THESE EXPRESSION PROFILES IN
THESE DIFFERENT DOMAINS ARE
CRITICAL FOR GIVING RISE OF
THESE TYPES OF INTERNEURONS BUT
WE HAD THE SINGLE CELL RNA DATA
AND WE GENERATE SINGLE CELL ATAC
DATA WHICH I WILL TOUCH ON LATER
SO WHAT WE DID IS -- THEY COME
FROM THE SAME REGIONS AND TIME
POINTS AND THIS ALLOWED US HOW
TO LOOK AT RNA AND CHROMATIN
ACCESSIBILITY CHANGES OVER TIME
AND IDENTIFY DIFFERENCES IN
BRAIN REGIONS.
SO WHAT WE SAW WAS IN INSTANCES
WHERE GENES WERE EXPRESSED
EARLIER IN THE NEUROGENIC
TIMELINE AND THEN WEPT DOWN OVER
TIME, WE SAW BLUE HERE IS THE
RNA, RED IS THE GAS AND THAT'S A
FANCY WAY OF SAYING PROMOTER
ACCESSIBILITY AND YOU CAN SEE IF
YOU FOLLOW THE BLUE HERE THE RNA
TURNS OFF PRIOR TO THE PROMOTER
AND THE ENHANCERS SHUTTING DOWN.
SO IT'S AS IF THE RNA IS DOWN
REGULATED BEFORE THE ACTUAL
PROMOTERS AND ENHANCERS TURNED
DOWN.
CONVERSELY IF YOU LOOK AT GENES
THAT ARE EXPRESSED LATER IN
DEVELOP AM, YOU SEE GREEN AND
RED THE PROMOTER AND ENHANCERS
BECOME ACCESSIBLE PRIOR TO THE
RNA BEING EXPRESSED.
AND THAT'S WHAT YOU WOULD
EXPECT, YOU NEED PROMOLT
PROMOTERS AND ENHANCERS OPEN
BEFORE YOU GET THE NEXT
EXPRESSION, SO THIS IS NICE, CAN
YOU DO IT AT MULTIO--METABOLIZD
AND 1 DATA AND 1 SELL, SO WE SEE
THESE 2 PATTERNS WHEN WE SEE
THESE DATA SETS INTEGRATED
TOGETHER.
NOW THIS IS GREAT IN LOOKING AT
RNA EXPRESSION, IT'S INFORMATIVE
BUT IT'S ONLY CORRELATIVE, BUT
WE CAN MAKE PREDICTIONS ABOUT
WHAT'S AN PROMOTER AND ENHANCER
BUT WE WANT TO HAVE GREATER DATA
SO I CAN SAY WITH MIER
CONFIDENCE, THIS IS AN ENHANCER
OF THIS GENE AND IT'S DIRECTLY
REGULATING ITS EXPRESSION EMPLOY
SO 1 WAY TO DO THAT IS LOOK AT
HISTONE MODISKS, SO HISTONES ARE
PROTEINS THAT DNA RAP WRAPS
AROUND THE HISTONES AND THESE
CAN BE MODJUSTIFIED IN A VARIETY
OF WAYS, PHOSPHORYLATED OR,
SETALATED AND DEPENDING ON THE
PATTERN OF MODIFICATIONS, IT CAN
BE INDICATIVE OF THE STATE OF
THE DNA.
SO IF WE LOOK AT 3 OF THESE
MARKS IN PARTICULAR, HK34 IS A
TRI-METHYLATION IS AN ACTIVE
PROMOTER, A3 KME4, AND THEN HK27
TRI METHYL, INDICATOR OF AN
CORRELATED PRESSIVE STATE.
SO WE CAN TAKE THESE AND
PERFORM MODERATIONS AND DO TAG,
SIMILAR TO CHPSEQ, BUT YOU NEED
LESS CELLS TO GET GOOD DATA AND
IT'S MUCH CHEAPER TO LOOK AT IN
BULK THESE METHYLATION PATTERNS
IN THESE DIFFERENT BRAIN
REGIONS.
SO HERE'S A SUMMARY OF HOW THIS
DATA CAN LOOK, SO WHAT WE HAVE
HERE IS AN ANALYSIS CALLED
SISSER O WHERE WE LOOK FOR
INDIVIDUAL CELLS, IF A LOCI IS
OPEN HERE AND THEN YOU GO DOWN,
DOWN STREAM OR UPSTREAM AND
THERE'S ANOTHER REGION HERE,
THAT IS OPEN IN THE SAME CELLS,
YOU KIND OF GET THESE PEAKS SO
THIS TYPE OF ANALYSIS IS
INTEREST, IF YOU'RE LOOKING AT A
PROMOTER AND YOU SEE A REGION
UPSTREAM OR DOWN STREAM THAT IS
OFTEN OPEN OR CO ACCESSIBLE IN
THE SAME CELL, THAT COULD BE
INDICIAIVE IT OF AN ENHANCER, SO
I FOCUS ON 1 GENE HERE CALLED
ASCL 1 AND THIS IS A GENE
EXPRESSIONED IN ALL THE
GANGLIONIC EMINENCES BUT IT'S
NOT EXPRESSED IN CORACLE CELLS
AND IN PREVIOUS WORK HAD ASSAYED
THIS REGION RIGHT HERE AS A
CANDIDATE ENHANCER FOR ASCL1.
NOW WHAT WE SEE IN OUR DATA IS
THAT IF YOU LOOK IN THESE 4
BRAIN REGIONS THIS LOCI IS
CERTAINLY ACCESSIBLE IN ALL THE
BRAIN REGIONS BUT IF YOU LOOK AT
THIS MARKER AND EFFECTIVE
ENHANCER IT'S STRONGER IN THE
CORTEX AND LESSER IN THE
GANGLIONIC INSTANCES AND AGAIN
THE BRAIN IS WHERE THE CORTEX IS
NOT EXPRESSED SO WHEN THEY LOOK
AT THIS, IT DID NOT DO A GOOD
JOB OF REPLICATING GENE
EXPRESSION AND WE WOULD ARGUE
BECAUSE OF DATA LIKE THIS THE
HIGHEST SIGNAL FOR AN ACTIVE
ENHANCER IS ACTUALLY IN CELL
TYPES WHERE THE GENE IS NOT
EXPRESSED.
CONVERSELY IF YOU LOOK DOWN HERE
IN GRAY, WE FOUND 2 OTHER
RENALLIONS WHERE YOU HAVE REALLY
HIGH SIGNAL FOR ACTIVE ENHANCERS
AND MUCH LOWER IN THE CORTEX SO
WE ARGUE THAT BY HAVING THIS
MULTIMODAL DATA SET, CAN YOU DO
A BETTER PREDICTION FOR
CANDIDATE ENHANCERS FOR VARIOUS
YEENS AND SO WE HAVE A WHOLE
LIST OF CANDIDATE ENHANCERS THAT
WE THINK COULD PLAY IMPORTANT
INSIGHT TO GENE REGULATION FOR
VARIOUS YEENS DURING
DEVELOPMENT.
SO THAT'S HIGHLIGHTED THERE.
SO ANOTHER ASSAY WE DID, SO FAR
WE HAVE ACCESSIBILITY, RNA AND
HISTONE MODIFICATIONS BUT IN
ORDER TO HAVE PROMOTER ENHANCER
INTERACTIONS, THE DNA NEEDS TO
COME INTO CONTACT.
AND SO TO REALLY LOOK AT THAT,
WE USED HIGH C OR MORE RECENTLY
MICROC IN COLLABORATION WITH
PEDRO [INDISCERNIBLE]'S LAB AT
NICHD AND SO WHAT HIGH C IS AN
ASSAY TO LOOK AT DIRECT DNA
CONTEXTS BETWEEN 2 DNA REGIONS.
AND SO, IF YOU JUST -- SO THIS
IS KIND OF A SCHEMATIC OF HOW
DNA CAN LOOK, BASIC LYE TO GET
ENHANCER AND PROMOTER TO COME
TOGETHER, THE DNA ACTUALLY HAS
QUITE ORGANIZED STRUCTURE, AND
THERE ARE VARIOUS WAYS TO BRING
THEM TOGETHER.
A LOT OF TIMES THEY INVOLVE THE
CTCF SITES THAT I WILL COME BACK
TO TOWARDS THE END OF MY TALK TO
KIND OF SEGGREGATE THE DNA IN
TOPOGRAPHICALLY ASSOCIATED
DOMAINS OR TABS AND THAT'S THE
WAY TO RESTRICT DNA FROM OTHER
REGIONS, AND ALLOW THESE
PROMOTER ENHANCER INTERACTIONS
TO OCCUR IN SPECIFIC WAYS.
AND SO BY USING THIS HIGH C
ASSAY, YOU CAN IDENTIFY THESE
LOCI WHERE YOU HAVE THE DISTAL
REGIONS COMING INTO CONTACT AND
PROVIDES GREATER INSIGHT TO HOW
THESE GENES ARE REGULATED.
SO THIS IS HOW THE HI-C LOOKS
LIKE.
ARE THESE HOT SPOTS OF VARIOUS
LOCI, AND IF YOU TAKE THE REGION
LIKE THIS, YOU FOLLOW IT DOWN
THE PYRAMID TO HEAR AND COME
COME DOWN THIS SIDE OF THE
PYRAMID AND THIS SIGNAL TELLS
YOU HAVE A DIRECT CONNECTION
BETWEEN THIS REGION HERE IN THE
DNA AND THIS REGION DOWN HERE SO
IF THIS HAPPENS TO BE A PROMOTER
OF A YEEN AND THIS IS A
CABBEDIDATE ENHANCER, THIS
PROVIDES FURTHER EVIDENCE THAT
YOU HAVE THIS GENETIC
INTEGRATION THAT YOU NEED.
SO WHAT WE DID IS WE PERFORM
THESE HI-C ASSAYS ON THESE 4
BRAIN REGIONS AND THEN WE LOOK
AT YEENS OF INTEREST TO FIND
REGIONS WHERE WE SAW
DIFFERENTIAL DNA INTERACTION, SO
HERE'S THE NK2.1 GENE THAT IS
EXPRESSED IN THE MG AND CRITICAL
FOR ALL CELLS DERIVE FRIDAY THE
MG AND HOPEFULLY CAN YOU
APPRECIATE THAT AT THE MGE, HAVE
YOU THE DARK INCREASE INTENSITY
HERE AND THAT'S ABSENT FROM
THESE OTHER BRAIN REGIONS, SO
THIS IS AN MGE SPECIFIC GENOMIC
INTERACTION.
NOW CAPTURE C IS KIND OF A
MODIFICATION OF HI-C AND IT
CONFIRMS THE DIRECT INTERACTION
BETWEEN IN THIS CASE, THE NK 2.1
PROMOTER AND A REGION DOWN
STREAM.
AND SO WHAT YOU SEE IS IN
INCREASED SIGNAL HERE WHICH
INDICATES THIS REAS YOJ HAS
DIRECT CONTACT WITH THE NK2.1
PROMOTER SPECIFIC TO THE MG, YOU
DON'T DO NOT SEE IT IN THE LG,
CG, CORTEX OR LIVERS OF CONTROL.
AND THEN YOU INDGREAT LITTLER
DATA WE HAVE WITH SINGLE CELL
RNASEQ, AND DIFFERENT
REGULATIONS AND REALLY TRULY
UNDERSTAND AT A GENOMIC LEVEL
AND CHROMATIN LEVEL BOTH HOW THE
CHROMATIN LOOKS AS 3D STRUCTURE
WHERE IT'S IRPT ACTIONS ARE, THE
HISTONE MODIFICATIONS TO TELL
YOU WHETHER SOMETHING IS A
LIKELY ENHANCER, PROMOTER OR
REPRESSED AND THEN ACCESSIBILITY
AND RNA AND WHAT I WILL NOTE
HERE IS THAT THIS MG SPECIFIC
INTERACTION OCCURS WITH THIS
GENE CALLED MBIP WHO HAS NOT --
WHOSE FUNCTION IS NOT KNOWN IN
NEURAL DEVELOPMENT AND IT'S
STUDIED IN CANCER BUT I DID FIND
A PAPER FROM ABOUT 15 YEARS AGO
WHERE THEY DID STAINING FOR MBIP
AND MBIP IS ALSO ONLY EXPRESSION
IN THE MG, IT'S NOT FOUND IN THE
OTHER BRAIN REGIONS.
SO NOW WE KNOW THERE'S A DIRECT
INTERACTION BETWEEN THESE 2
GENES THAT ARE ONLY EXPRESSED IN
THE MG AND NOT FOUND IN OTHER
BRAIN REGIONS.
AND THEN WE WENT AHEAD AND YOU
CAN DO THIS FOR OTHER GENES AND
OTHER BRAIN REGIONS, AND THERE'S
LOTS OF KIND OF EXCITING INSIGHT
YOU HAVE, WE HAVE INTO REGION
SPECIFIC BRAIN REGION SPECIFIC
CHROMATIN INTERACTIONS, IN THE
EMBRYONIC BRAIN AND TO OUR
KNOWLEDGE THIS HASN'T BEEN
EXPLORED IN DETAIL YET.
SO WE REALLY THINK, WE
UNDERSTAND THE GROUND TRUTH OF
HOW THESE CELLS LOOK IN THE
EMBRYO AS WE'RE MAKING THESE
CRITICAL FATE DECISIONS.
NOW I WILL HIGHLIGHT 1 ONGOING
STUDY THAT CAME OUT OF THIS
SPECIFIC NK2.1 INTERACTION.
AS I MENTIONED MASTER REGULATOR
OF CELL FATE AND SO WE WANTED TO
KNOW HOW CRITICAL IS THIS IRPT
ACTION FOR EXPRESS AND FUNCTION
OF THESE YEENS?
IS THERE A WAY TO PERTUSH THIS
INTERACTION TO DISRUPT IT AND
SEE HOW THAT AFFECTS GENE
EXPRESSION CELL FATE AND WHY
THIS IS ALSO RELEVANT IS BECAUSE
IN HUMAN DISEASE, THERE ARE
VARIANTS OF NKX 2.1 YEEN AND IT
GIVES RISE TO COMPLEX SYNDROME
THAT CAN BE CALLED BENIGN
HEREDITARY CHOREA, OR BRAIN LUNG
THYROID SYNDROME, IT'S ALSO
EXPRESSED IN THE LUNG AND
THYROID.
SO PATIENTS THAT HAVE MUTATIONS
AND VARIANTS OF THE NK2.1 CAN
EXPRESS PHENOTYPES THAT SHOW
DYSFUNCTION IN THESE 3 ORGANS
WITH VARIOUS SEVERITYS DEPENDING
ON THE PATIENT.
NOW WHAT'S ALSO REALLY
INTERESTING IS THAT A HANDFUL OF
PATIENTS THAT PRESENT WITH THIS
PHENOTYPE DO NOT ACTUALLY HAVE
MUTATIONS IN NK2.1.
RATHER THEY HAVE DELETIONS
FURTHER UPSTREAM THAT ENCOMPASS
THE MBIP GENE AND THERE ARE NO
KNOWN FUNCTIONS FOR THIS
NEURODEVELOPMENT.
SO NOW WE UNDERSTAND THIS
INTERACTION HERE WE WONDERED IF
IT'S POSSIBLE THAT SOME OF THIS
PHENOTYPE THAT ARISES IN THESE
PATIENTS WERE THE NK2.1 LOCI IS
INTACT BUT THE MBIP IS DISTURBED
OR MUTATED COULD BE DUE TO THIS
CRITICAL INTERACTION HERE, SO
AGAIN, HOW DO WE STUDY THIS, HOW
DO WE BREAK UP THIS
INTERACTION,.
>> MUNICIPAL ADVISERS AND I
ASK'S QUESTION?
>> YES.
>> HOW IS THE GENE PRODUCTION
WITH THIS AND [INDISCERNIBLE]?
>> YEAH, GOOD QUESTION AND KIND
OF COMPLICATED.
SO AT THIS RESOLUTION WITH WE
DID THIS DATA AT HI-C WE DID NOT
HAVE THE RESOLUTION TO KNOW
WHETHER THE INTERACTION, SO THAT
YOU KNOW, ALL I CAN SAY HERE IS
THIS INTERACTION IS SOMEWHERE IN
THIS MBIP LOCUST, YOU CAN SEE
THIS IS A GENE WITH 5 OR 6 AXONS
AND THE RESOLUTION WE HAD IS WE
KNOW THERE'S AN INTERACTION
SOMEWHERE THERE WHETHER IT'S A
PROMOTER-PROMOTER INTERACTION OR
WHETHER THERE'S A PARTICULAR
LOCI WITHIN AN MBIP ENTRON,
THAT'S AN ENHANCER FOR NK2.1 OR
VICE VERSA, WE CAN'T SAY
ANYTHING ABOUT IT AT THIS LEVEL
OF RESOLUTION.
I MY KROSIA I TALK ABOUT A
LITTLE BIT DOES GIVE YOU BETTER
RESOLUTION WHERE YOU CAN LOOK AT
10S OF NUCLEOTIDES AND SO THEN
WE CAN SAY WITH CONFIDENCE THAT
THERE'S INTERACTION BETWEEN THE
NK2.1 PROMOTER AND SOME NEUTRON
WEAN IT AND MBIP BUT THERE'S
PROMOTER-PROMOTER INTERACTIONS
THAT HELP BOTH GENES AND THEN
THERE'S ALSO EVIDENCE FOR
ENHANCERS OR ENTRON NEAR ANOTHER
GENE AFFECTING THE GENE
EXPRESSION OF ANOTHER 1.
SO THAT'S REALLY A QUESTION OF
RESOLUTION, HOW REFINED CAN YOU
GET TO LOOK AT THIS INTERACTION.
>> [INAUDIBLE QUESTION FROM
AUDIENCE ]
>> YEAH, AGAIN ABOUT RESOLUTION.
ALL WE CAN SAY ABOUT THIS IS
THERE'S A DIRECT INTERACTION
BETWEEN SOME REGION HERE IN THE
MBIP YEEN AND THE IN, K2.1
PROMOTER.
IT'S POSSIBLE THERE ARE 2 OR 3
DISTINCT INTERACTION DOMAINS BUT
WE DON'T HAVE THE RESOLUTION
WITH THIS ASSAY TO DIRECTLY
INTERACT WITH THAT.
>> [INAUDIBLE QUESTION FROM
AUDIENCE ]
>> SO CAN YOU LOOK AT
INTERCHROMESOME INTERACTIONS
WITH HI-C.
SO WHAT I'VE DONE HERE IS FOCUS
ON THE SPECIFIC REGION BUT CAN
YOU EXPAND THIS OUT TO ALL
CHROMATIN -- CHROMESOME
INTERACTION SO IF THERE ARE IRPT
ACTIONS BETWEEN CHROMESOMES THAT
OCCUR AT A HIGH ENOUGH PREQUENCY
THAT IT WOULD KIND OF GENERATE
THESE HOT SPOTS YOU COULD
THEORETICALLY DETECT IT IN THIS
TYPE OF DATA.
BUT IN THIS CASE I FOCUSED ON
THIS LOCI OF INTEREST WHERE WE
HAVE THIS GENE AND THEN A STRONG
BRAIN REGION SIGNAL JUST
UPSTREAM, OKAY IN OKAY, SO HOW
CAN WE DISRUPT THIS INTERACTION?
THIS IS WHERE OUR COLLABORATION
WITH PEDRO RO, KREBS CYCLE CO
WAS CRITICAL, SO, IN A STUDY
THAT WE COLLABORATED WITH, HE
WAS LOOKING AT THE SOX 2 LOCUST
WHICH IS A CRITICAL GENE OF
INTERACTION FOR THE EPIBLAST AND
SO BASICALLY YOU HAVE A CRITICAL
ENHANCER OF SOX 2 WHICH IS
CALLED SCR OVER HERE AND THEN
THE SOX 2 GENE IS OVER HERE.
AND YOU HAVE THESE LITTLE ARROWS
ARE CALLED CTCF SITES AND AS I
MENTIONED THAT'S CRITICAL FOR
PARSING THIS GENOME STRUCTURES
THAT YOU HAVE THIS TAD DOMAIN
HERE SO THAT BASICALLY GENOMEEC
LOCI HERE CAN INTERACT WITH EACH
OTHER AND REALLY ARE INHIBITED
FROM INTERACTING OUT HERE.
SO PEDRO'S IDEA IS WHAT IF WE
PUT IN ARTIFICIAL CTF SITES HERE
TO DISRUPT THE INTERACTION AND
THAT'S WHATEE DID IN THIS PAPER,
CAN YOU DO DIFFERENT KIND OF
TYPES OR DIFFERENT ORGANIZATIONS
OF CTCF FACTORS AND I HOPE YOU
CAN APPRECIATE THAT WHEN YOU DO
THESE MODIIVESS WHAT YOU DO IS
DISRUPT OR IN THIS CASE MINIMIZE
THIS INTERACTION BETWEEN THE SOX
2 LOCUST AND THE ENHANCER OVER
HERE AND THEN CAN YOU LOOK AT
HOW THIS AFFECTS GENE EXPRESSION
PHENOTYPE.
SO WE SAID GREAT, CAN WE DO THIS
AT THE LOCI WE CARE ABOUT IN THE
NK2.1 PROMOTER?
AND LUCKILY IT HAD THE SITES SET
UP IN A WAY THAT ALLOWED US TO
DO THIS SO PEDRO DESIGNED
EXOGENOUS CTCF SITES TO INSERT
ON IN THESE 2 YEENS WITH THE
GOAL OF BLOCKING THE
INTERACTION.
THIS IS NEW DATA WITH MY ROUGH
ATOM SEEDS, WITH HIGH
REZONINGUTION AT HI-C AND YOU
HAVE THE NK2.1 YEEN HERE AND
THEN YOU WILD-TYPE HERE IN THE
INTERACTION IN THE MICE WHERE WE
INSERTED THE CTCF SITES, YOU SEE
THE INTERACTION IS GONE AND
INSTEAD YOU HAVE AN ISHT
INTERACTION HERE WHERE YOU HAVE
THE BLUE CTCF SITE, INTERACTING
WITH THESE RED SITES, SO THIS
WAS SUCCESSFUL IN PERTURBING
THIS INTERACTION BETWEEN THESE 2
LOCI.
AGAIN, CAPTURE MICRO-C, ANOTHER
WAY OF LOOKING AT IT, YOU SEE
THE WILD-TYPE, IT'S NOW REDUCED
IN THE MUTANT WITH AN ARTIFICIAL
IRPT ACTION HERE SO WE CONFIRM
THAT BASICALLY WE BLOCK THIS
NK2.1 MBIP INTERACTION IN THESE
MICE.
>> QUESTION [INAUDIBLE QUESTION
FROM AUDIENCE ]
>> SO THIS IS 3, WE HAVE 3 GOING
THIS WAY AND 3 GOING THIS WAY,
AS KIND OF THE MORE CTCFs
SITES YOU IN TERMS OF BLOCKING
THAT, THESE ARE HUMAN SITES SO
THESE MAKES IT EASIER FOR
SEQUENCING AND DOWN STREAMS
OKAY, SO WHAT HAPPENS IN GENE
EXPRESSION, THIS IS INSITUS
LOOKING AT NK2.1 AND MBIP AND
YOU CAN SEE IN THE MUTANT IS
THAT THE MBIP EXPRESSION IS
ALMOST TOTALLY GONE IN THE NK
AND CTCF INSERTION, IT BEING
LOAMACYS LIKE THERE'S DOWN
REGULATION OF NK2.1 PRACTICES
FORMING ADDITIONAL CAN FIRM
THIS, BUT IF YOU LOOK AT THE PC
R, YOU SEE STRONG REDUCTION, 50%
OR SO REDUCTION OF MBIP IN THIS
MUTANT, IT LOOKS STRONGER BY
INSITUS AND WE SEE A SLIGHT BUT
PROBABLY SIGNIFICANT DECREASE IN
MK2.1.
AND WE THINK THIS IS EXCITING
BECAUSE AGAIN GOING BACK TO
THOSE PATIENTS THIS COULD
INDICATE THAT IN THESE PATIENTS
WHERE YOU HAVE THE LOCUST, WHAT
REALLY COULD BE HAPPENING IS
THAT YOU'RE DISRUPTING THIS
PARTICULAR INTERACTION THAT'S
DECREASING GENE EXPRESSION AND
PROBABLY PERTURBING NK2.1
EXPRESSION EVEN THOUGH THE
NKSUPER 1 LOCUST IS IN TACT AND
THUS GIVING RISE TO THE
PHENOTYPE.
SO WE'RE KIND OF WORKING ON
THESE MICE, IT CHANGES IN CELL
FATE AND ANY TYPE OF BEHAVIORIAL
DEFICITS BUT HERE'S A NICE BRAIN
REGION SPECIFIC INTERACTION WE
IDENTIFIED AND WHEN YOU
MANIPULATE THAT, THIS
INTERACTION NOT THE GENES
THEMSELVES, CAN YOU SEE CHANGES
IN JEN EXPRESSION AND HOPEFULLY
CELL FATE.
>> [INAUDIBLE QUESTION FROM
AUDIENCE ] -- SO IT LOOKS
REALLY, REALLY STRAIMPLEG TO --
STRANGE TO ME, WHAT YOU ARE
DOING HERE DISRUPTING SUPPOSELY
EKING LATTERY ELEMENT LOCATED
INSIDE MBIP, YOU ARE SEARCHING A
BLOCK BETWEEN THAT ELEMENT AND
NKX2.1?
AND YOU DO SEE DECREASE LEVEL OF
EXPRESSION IN NKX2.1, GREAT, BUT
YOU'RE NOT REMOVING THAT
ELMETROPOLITAN AWAY FROM THE
HOST MBIP GENE AND YET THE LEVEL
OF EXPRESSION DRIVES DOWN
SIGNIFICANTLY.
ARE YOU SAYING THAT NKX2.1 IN
TURN UPREGULATES MBIP OR --
>> THIS GOES BACK TO YOUR
PREVIOUS QUESTION, IS THIS A
PROMOTER PROTEIN COMPLEXER
INTERACTION OR PROMOTER
INTERACTION WHERE THEY'RE
HELPING EACH OTHER.
I CAN'T SAY DIRECTLY WHAT THE
WORRY IS.
BASED ON THIS, THESE ARE 2
PROMOTERS COMING TOGETHER THAT
ARE HELPING EACH OTHER
UPREGULATE THEIR EXPRESSION AND
NOW WE FORCE SEPARATION OF THIS
AND FOR WHATEVER REASON MBIP IS
STRONGLY REDUCING NKX2 PBT 1,
AND AS I MENTIONED, NKX2.1 IS A
CRITICAL REGULATORS FROM ALL
CELLS FROM THE MBIP, SO MY GUESS
IS THERE'S A MORE EVOLUTIONARY
CONSERVATION THERE THAT IS
CRITICAL FOR KEEPING NK2.1 ON SO
I DIDN'T MENTION IT, I'LL HOP
BACK REAL QUICK BUT IN THAT SOX
2 LOCUST STUDY, YOU SEE HERE IS
THAT DESPITE THE BEST EFFORTS
THERE'S STILL ACTUALLY AN
INTERACTION HERE DESPITE PUTTING
IN THESE SITES.
WHAT THIS PAPER SHOWED IS THAT
IT SEEMS LIKE EVOLUTION HAS
FOUND A WAY AROUND VARIOUS
PROTURBATIONS LIKE THIS BECAUSE
SOX 2 IS SO CRITICAL FOR
DEVELOPMENT THAT ANY LITTLE
MUTATION HERE OR THERE, THERE'S
MULTIPLE MECHANISMS TO HELP
THESE INTERACTIONS COME TOGETHER
SO THAT SOX 2 EXPRESSION IS
FOCUSED AND I WOULD ARGUE THAT
MAYBE THE SAME THING IS
HAPPENING AT NK2.1 BECAUSE IT'S
SO CRITICAL FOR BRAIN
DEVELOPMENT, LUNG DEVELOPMENT,
THYROID DEPRIVATIONMENT THAT
THERE ARE YEENS THAT HAVE THIS
EVOLUTIONARY CONSERVED MECHANISM
THAT IF EVEN WE TRY TO PERSH
TURB THEM WITH OTHER
INTERACTIONS THEY HAVE OTHER
ENHANCERS OR OTHER WAYS TO TRY
TO KEEP EXPRESSION OF THE GENE
AT CRITICAL TIMES.
THAT'S THE BEST ANSWER I HAVE.
BUT I THINK THAT IS A PROMOTER,
PROMOTER INTERACTION RATHER THAN
AN EN--STRATEGIES HANSER
PROMOTER BUT AGAIN, WE DON'T
HAVE GREAT EVIDENT FOR THAT 1
WAY OR THE OTHER.
>> OKAY, SO, THIS KIND OF WRAPS
UP THE FIRST PART OF THE STORY
WHERE I'VE SHOWN YOU HOW WE
GENERATE AN EPIGENOME ATLAS IN
THE EMBRYONIC MOUSE BRAIN WITH A
VARIETY OF DATA SO THAT WE TRULY
UNDERSTAND HOW THESE CELLS LOOK
BOTH AT AN INDIVIDUAL CELL
LEVEL, AND A GLOBAL CHROME TIN
LEVEL.
WE'VE IDENTIFIED MANY, WHAT I
WOULD CALL HIGH CONFIDENCE
ENHANCERS FOR THAT QUITE
POSSIBLY OR CRITICAL FOR
REGULATION OF GENES THAT ARE
IMPORTANT FOR DEVELOPMENT OF
CELLS IN THESE BRAIN REGIONS.
IT'S IMPORTANT FOR ME TO MAKE
THIS DAT AS PUBLICLY AVAILABLE
AS POSSIBLE, SO IN ADDITION TO
ALL THE SEQUENCING BEING
DEPOSITED WE USED UCSD BROWSER
SO ANYBODY CAN GO TO THE WEBSITE
AND SEARCH FOR THEIR FAVORITE
GENE AND LOOK FOR MODIFICATION,
HISTONE DEVELOPMENT AND WE THINK
IT'S A VERY USEFUL TOOL FOR
STUDYING BRAIN DEVELOPMENT AND I
GAVE EXAMPLES OF HOW PERTURBING
THEM CAN LEAD TO INSIGHTS AND
MECHANISMS THAT POSSIBLY HAVE A
ROLE IN HUMAN DISEASE.
OKAY, SO NOW THAT WE'VE KIND OF
DEVELOPED THIS KNOWLEDGE AND
THESE STRATEGIES, WE'RE PRETTY
WELL POSITIONED TO UNDERSTAND
HOW THESE ASSPEBTS OF GENES OR
GENE REGULATORY MECH NIMS CAN
AFFECT CELL FATE AND DISEASE, SO
WE'VE BASICALLY KIND OF BEEN
GOING THROUGH THIS TYPE OF
PIPELINE TO ARK ASSESSES THIS
REWRILIENCE WE HAVE A VERY
MUTATION, PROTURBATION, WE CAN
LOOK AT CHROME TIN ORGANIZATION
AND TRANSCRIPT ORDER OF
MICRONSAL WILL SINGLE CELL
LEVEL, WE CAN ASSAY CELL FATE,
WE CAN LOOK AT CELL FUNCTION
WITH THE COLLABORATORS
PERFORMING ELECTROPHYSIOLOGICAL
EXPERIMENTS AND THEN ULTIMATELY
ANIMAL BEHAVIOR AND I WILL SHOW
YOU HOW THESE AFFECT ANIMAL
BEHAVIOR AND HOW IT MIGHT RELATE
TO HUMAN TEASE AND SO THERE'S 3
STORIES DEPENDING ON TIME, I
WILL SEE HOW MUCH I CAN GET
THROUGH.
THE FIRST 1 IS WHERE WE LOOK
THEA PROTURBATION OF YEEN
REPRESSION USING LOSS OF ACH2
AND THEN ACTIVATION AND THEN
DISEASE, SO WE'RE FESTER TAKING
A SLEDGE HAMMER TO THE WHOLE
SYSTEM.
WE KNOW WHAT'S UPON HAING NOW IF
WE TRY TO DISRUPT GENERAL GENE
REPRESSION OR GENERAL GENE
ACTIVATION SPECIFICALLY IN THE
MG OF THESE MICE.
AND I WILL GIVE BACKGROUND ON
ECH 2, THIS IS A CRITICAL
METHYLTRANSFERASE, THIS IS A BIG
COMPLEX BUT WHAT'S IMPORTANT TO
KNOW IS THAT IT'S CRITICAL FOR
METHALATING K3 H3 K27 AND HAD IS
IMPORTANT FOR GENE REPRESSION.
SO BY BLOCKING OR PERTURBING ANY
COMPLEX, YOU WILL DISRUPT THE
NORMAL GENE REPRESSION THAT WILL
LEAD TO ABERRANT GENE
PROTURBATION.
NOW WHY THIS IS IMPORTANT
BECAUSE OF A NUMBER OF STUDIES
THAT LOOK AT EZH2 IN THE BRAIN
DEVELOPMENT AND WE SEE THAT LOSS
OF EZH2 CAN CHANGE THE LOSS OF
FUNCTION, MIGRATION PATTERNS,
AND THE ROLE THAT WAS STUDIED IN
BRAIN DEVELOPMENT BUT NOT
PARTICULARLY IN THE FOREBRAIN
NUROONS AND BECAUSE THESE CELLS
UNDERGO COMPLEX BEHAVIOR,
CRITICAL FOR CELL FATE WE
THOUGHT THIS WAS A NICE TARGET
TO DISRUPT GENE REPRESSION IN
THE MGE AND LOOK AT HOW THAT
SLEDGE HAMMER APPROACH AFFECTS
CELL FATE.
SO WE USE AN NPXCREE TARGET AND
THIS IS TARGETED TO THE MGE WITH
THE FLOX EZH2, AND YOU SEE THE
NORMAL EXPRESSION AND THEN THE
BEING IMO OUT THAT EXPRESSION IS
STRONGLY DOWN REGULATED
SPECIFICALLY IN THE MG BUT
INTACT IN THE LG AND CORTEX.
THE READ OUT FOR THIS YEEN IS
CRITICAL FOR METHALATING THE H3
K RESIDUE AND WE SEE A DOWN
REGULATION OF THIS HISTONE
MODIFICATION.
SO WHAT WE DID IS WE TOOK THE
MICE, HARVESTED THE BRAINS IN
THE ADULT AND WE COULD PERFORM
AMINO STAINING SO SST AND PV ARE
MARKERS FOR 2 SUBTYPES THAT,A
RISE FROM THE NG, BASICALLY 95%
OF THE CELLS IN THE CORTEX THAT
ARE BORN IN THE MG EXPRESS
EITHER SST OR PV, SO THESE ARE
NONOVERLAPPING SUBGROUPS THAT
ENCOMPASS ALL CELLS IN THE
CORTEX, SO WHAT YOU SEE IN THE
KNOCK OUT HERE IS WE SEE AN
OVERALL DECREASE IN THE NUMBER
OF TOMATO CELLS, TOMATO IS A
MARKER FOR ALL MG CELLS, THIS IS
JUST A REPORTER SO MAYBE CAN YOU
TELL HERE BUT THERE'S ABOUT A
20% REDUCTION OR SO OF ALL MG
CELLS IN THE CORTEX BUT WE
ACTUALLY SAW A DIFFERENT AFFECT
IN CELL FATE.
IF YOU LOOK AT SST IN GREEN HERE
THERE'S AN INCREASE IN THE
AMOUNT OF SST INTERNONS AND
CORRESPONDING DECREASE IN THE PV
CELLS.
SO THIS FEWER CELLS DUE TO THIS
MUTATION SPECIFICALLY IN THE
MGE.
I WON'T SHOW YOU THE DATA.
THERE'S ALSO INTERNEURONS THAT
ARE IN THE STRIATUM AND
HIPPOCAMPUS AND WE SAW MORE FATE
SWITCH WHERE WE SAW MORE PV AND
SST CELLS.
I WILL NOTE THESE ARE CYCLING
CELLS, WE USE A DIFFERENT CREE
LINE TO KNOCK OUT POST MITOTIC
CELLS SO AFTER THESE CELLS ARE
BORN AND WE DO NOT SEE THESE
CHANGES IN CELL FATE.
SO THIS TELLS US THE FUNCTION OF
ECH2 IS CRITICAL, AND ONCE THEY
BECOME POST MITOTIC AND REMOVING
THOSE IN THE CELLS DOES NOT
CHANGE CELL FATE.
SO WITH OUR COLLEAGUES
[INDISCERNIBLE] AT NIMH, THEY
PERFORMED ELECTROPHYSIOLOGY
RECORDINGS TO SEE IF IN THESE
MUTANTS IS THE PHYSIOLOGY
AFFAIRS TEAM LEADERRERRED?
SO THEY LOOKED AT ABOUT 20 OF
THESE CELL ANDS THERE WAS VERY,
VERY LITTLE DIFFERENCES BUT WHAT
THEY DID NOTICE, STRIKING
CHANGES IN THE MORPHOLOGY, SO
THESE ARE BARK LBUMIN, AND
THERE'S MORE BRANCH XG THERE'S
QUANTIFIED OVER HERE SO WHAT
THIS MEANS THAT IN THIS MOUSE
LINE WHERE YOU HAVE FEWER PV
CELLS, THE PV CELLS REMAIN HAVE
GREATER AXONAL PROJECTIONS AND
GREATER TARGETS AND PRESUMABLY
GREATER SYNAPSES, SO WE'VE
EITHER SINCE THE COMPENSATION
MECHANISM, HOW DOES THE BRAIN
COMPENSATE FOR FUHRER INHIBITORY
NEURONS AND THE WAY TO DO IS
THAT THE 1S THAT SURVIVE, THEY
HAVE MORE PROCESSES, CONTACT
MORE CELLS TO TRY TO INCREASE
INHIBITION.
AND THEN WHAT WE WANT TO DO IS
PERFORM MULTIOHM ANALYSIS SO IN
ADDITION TO LOOKING AT CHANGES
IN CELL FATE WE CAN UNDERSTAND
WHAT'S GOING ON GENETICALLY AND
GENE REGULATORY MECHANISMS IN
THESE CELLS, AT THIS POINT
MULTIOHM WAS AVAILABLE, SO THIS
IS SINGLE CELL ATAC AND RNA IN
THE SAME CELL SO WE PERFORM
MULTIOHM ANALYSIS, KNOCKOUT MICE
ON 2 DIFFERENT EMBRYONIC AGES.
AGAIN WE CAN KIND OF LOOK AT
GENE EXPRESSION PROFILES SO WE
KNOW THE TRANSLATIONAL -- TRAJO
MOST MITOTIC CELLS ARE NEAR THE
BOTTOM AND WE WANTED TO SEE, ARE
THERE SIGNATURES IN THE EMBRYO
THAT ARE INDICATIVE OR CAUSATIVE
FOR THESE CHANGES IN CELL FATE.
SO IF WE LOOK AT SST HERE, AND
AGAIN, THIS IS -- THERE'S MORE
SST CELLS IN THE ADULT MOUSE, WE
SEE AN INCREASE IN SST
EXPRESSION HERE IN THE KNOCK OUT
MOUSE, AT E15, COMPARED TO THE
WILD-TYPE.
CONVERSELY, PARVE O BIEWMIN IS
NOT EXPRESSED, IT DOESN'T COME
ON UNTIL POSTINATEALLY, BUT
THERE ARE A COUPLE GENES MAP 2 C
AND MAP B, THAT ARE PREDICTIVE
OF THE WILD-TYPE CELLS.
YOU CAN SEE HERE WE HAVE STRONG
EXPRESSION OF THE CELLS AND IN
THE KNOCK OUT YOU SEE DOWN
REGULATION OF THESE.
IN ADDITION BECAUSE IT'S
MULTIOHM, WE CAN LOOK AT THE
MOTIF FOR THE BINDING SITE OF
THESE FACTORS IN OUR DNA, SORRY
IN OUR CELLS AND YOU CAN SEE
AGAIN, A DECREASE IN THE MOTIF
ACCESSIBILITY IN THE KNOCK OUTS
COMPARED TO THE WILD-TYPE.
SO NOW WE CAN IDENTIFY IN THE
EMBRYO, SOME GENETIC LOGIC AS TO
WHY WE SEE THE CELL FATE CHANGES
INCREASED IN MET STATIN IN D15
AND DECREASED MARKERS OF PV
CELLS ALREADY PRESENT ALSO AT
E15.
WE CAN DO KIND OF UNBIASED
COMPARISON TO LOOK AT GENES THAT
ARE UPREGULATED AT THESE
DIFFERENT AGES IN THESE VOLCANO
PLOTS AND AGAIN YOU SEE 2 GENES
HERE PREDICTED OF PV CELLS THAT
ARE WILD-TYPE ENRICHED MEANING
DOWN REGULATED OF THE KNOCK OUT
AND SST OVER HERE IS STRONGLY
ENRICHED IN THE KNOCK OUT.
THERE'S A FAIR AMOUNT OF OTHER
GENETIC ANALYSIS WE'VE DONE WITH
THIS BUT I DON'T WANT TO BELABOR
THE POINT TOO MUCH IS BASICALLY
THE TAKE HOME MESSAGE IS THAT WE
SEE A FATE SWITCH IN THIS MOUSE,
AND ALSO, WE SEE CHANGES IN GENE
EXPRESSION IN THE EMBRYO THAT
ARE CERTAINLY CAUSATIVE FOR THAT
FATE SWITCH.
OKAY, NOW I'M GOING TO JUMP TO
ANOTHER STORY, SO THAT WAS
LOOKING AT GENE REPRESSION, AND
SO THIS IS -- SORRY, I FORGOT I
HAD THIS SLIDE.
SO IN ARK DITION TO THE MULTIOHM
DATA WE ALSO PERFORM TAG TO
LOOK THEA DIFFERENCES OF THIS
KTWEIVE METHYLATION, AGAIN THIS
IS THE TARGET OF ECH2, AND THE
TAKE HOME MESSAGE WE FOUND THAT
IN THIS KNOCK OUT THE CHANGES IN
THE K27 METHYLATION WAS NOT
EQUIVALENT ACROSS THE GENOME.
WE SAW SOME LOCI THAT WERE MORE
SUSCEPTIBLE SUCH AS THE FOX
PPAYS LOCI HERE, YOU SEE THE
WILD-TYPE AND YOU SEE A REALLY
STRONG REDUCTION IN MULTIPLE
REPLICATES IN THE KNOCK OUT.
SO THIS IS A LOCI THAT SEEMS TO
BE EXTREMELY SUSCEPTIBLE TO THIS
MANIPULATION.
CONVERSELY, NKX2.1, YOU CAN SEE
A RELATIVE INCREASE IN THE LEVEL
OF METHYLATION, SO THIS DOESN'T
MEAN IT'S MORE METHALATED THAN
THE WILD-TYPE, IT JUST MEANS
IT'S MORE RESISTANT, THE
METHYLATION WAS NOT REMOVED
BECAUSE ALL OF THIS IS RELATIVE.
SO THIS KIND OF GOES BACK TO
YOUR EARLIER POINT, I THINK
THERE'S EVIDENCE THAT NK2.1 HAS
SOME SORT OF -- BECAUSE OF ITS
CRITICAL ROLE IN FATE, I THINK
THERE'S EVOLUTIONARY
CONSERVATION THAT HAS CAUSED
THIS LOCI TO BE MORE RESISTANT
TO VARIOUS CHANGES BECAUSE
DESPITE KNOCKING OUT A GENE FRM
RETIRED KRAL METHYLATION, IT
TRIES AS BEST IT CAN TO REFUTE
THIS PROTURBATION AND KEEP HIGH
LEVELS OF METHYLATION HERE, SO
THAT IT CAN BE REPRESSED OR
ACTIVATED IN THE PROPER WAY.
>> OKAY, SO THAT WAS YEEN
REPRESSION AND NOW I WANT TO
TALK A LITTLE BIT ABOUT GENE
ACTIVATION BUT ALSO WHY THIS
PARTICULAR MODE OF LOOKING AT
GENE ACTIVATION IS CRITICAL.
SO METHYLATION AT H3 K4, IS
CRITICAL FOR GENE ACTIVATION,
BUT THERE'S A LOT OF EVIDENCE
THROUGH YEARS OF WORK THAT HAVE
SHOWN THAT THIS REGULATION OF
THIS LOCI AND THIS METHYLATION
IS ASSOCIATED WITH A VARIETY OF
NEURAL DEVELOPMENTAL DECS,
SCHIZOPHRENIA, ALSO, DRUG
DEPENDENCY, THIS IS A NICE
SUMMARY OF A HOST OF YEENS THAT
ARE EPIGENETIC MACHINERY THAT
CAN EITHER WRITE OR REMOVE
EPIGENETIC MARKS AND I WILL
HIGHLIGHT 3 GENES HERE, ALL 3 OF
THESE YEENS TARGET METHYLATION
OF THIS PARTICULAR RESIDUE AND
ALL HAVE VARIOUS SYMPTOMS AND
DRIVE HUMAN DISEASE.
SO WE THINK A BETTER
UNDERSTANDING OF HOW THIS
PARTICULAR LOCI, GETS METHALATED
IT'S CRITICAL FOR UNDERSTANDING
GENE ACTIVATION AS WELL AS MAYBE
KIND OF THE UNDERLYING CAUSES OF
VARIOUS HUMAN DISEASES.
NOW WHAT MAKES STUDYING THIS
COMPLICATE SIDE THAT UNLIKE THE
PREVIOUS EXAMPLE WHERE IT'S THE
PRIMARY METHYLTRANSFERASE THAT
TARGETS THAT HISTONE, IN THIS
CASE YOU HAVE 6 DIFFERENT
ENZYMES THAT CAN METHALATE K4,
THAT CAN METHALATE THE K4
RESIDUE AND THESE ARE EXPRESSED
OFTEN TIMES IN OVERLAPPING CELLS
AND CAN OVERLAPPING COMPENSATORY
FUNCTION SO SIMPLY KNOCKING OUT
1 OF THESE IS SOMETIMES NOT
SUFFICIENT TO HAVE A ROBUST
EFFECT.
NOW WE WERE LUCKY THAT A
COLLEAGUE HERE AT NIDDK, DR. GUY
HAD DEVELOPED A MOUSE WHERE HE
MUTATED THE SLICE IN 4 RESIDUE
TO A AN M METHYL, I FORGET, K4 M
AND BASICALLY WHAT THIS DOES IS
BLOCKS THE LIESING, BECAUSE IT
CAN NEVER BE LIESINE BECAUSE
IT'S NEVER THERE ANYMORE AND
IT'S CREE DEPENDENT WHICH,A
LOWSITOUS EXPRESS IN MUTATED
HISTONE IN THE CELLS WE WANT AND
WHAT'S NICE IS THAT THIS IS
TRANSGENIC SO BASICALLY YOU HAVE
IN A K-FORM HOMOZYGOUS MOUSE,
YOU HAVE 2 WILD-TYPE ARK LEGALS,
AND ALSO ELSEWHERE WHERE THIS
WAS INSERTED YOU HAVE 2 MUTANT
ALLELES.
SO THIS IS MIMICS HUMAN DISEASE
WHERE YOU HAVE A KNOCK DOWN OR
MUTATION IN AN ENZYME THAT
AFFECTS THIS AND YOU'RE LOOKING
AT A LOSS, NOT A COMPLETE LOSS
OF FUNCTION BUT A IMK DOWN.
SO WE THINK THIS IS A COOL TOOL
TO BASICALLY HAVE A HOMOZYGOUS
MOUSE AS A FUNCTIONAL HAT TO
STUDY MANIPULATION OF THIS
RESIDUE.
SO WHAT WE SEE OUT OF THESE
HOMOZYGOUS MOUSE, THEY'RE
ACTUALLY UNDERWEIGHT EARLY ON, A
NUMBER OF THEM DIE, 50% OF THEM
BY BY 20 WEEKS, THEIR BRAINS ARE
ALSO SMALLER WHICH YOU CAN SEE
HERE, THE CORTEX HAS DECREASED
THICKNESS, BUT WHAT'S
INTERESTING IS THAT THE 1S THAT
SURVIVE AFTER ABOUT 12 MONTHS
BECOME QUITE OBESE, THEY CAN BE
DOUBLE THE WEIGHT OF THEIR
COUNTERPARTS AND AGAIN, I WON'T
GO THROUGH THIS IN MUCH DETAIL
BUT WE ARE FAIRLY CONFIDENT THIS
IS PROTURBATIONS IN THE
HYPOTHALAMUS, AND HYPOTHAT
WILLUS IS CRITICAL FOR FEEDING
BEHOUSTON AND RICE FOOTBALL AND
VARIETY OF OTHER HORMONAL
BEHAVIORS.
NOW IF YOU LOOK AT CHANGES IN
THE INTERNEURON COUNTS, WE SEE
THERE'S A DECREASE IN MEAS MICE
IN THE NEURONS AND A DEC --
STRONG DECREASE IN P A LBALBUT
MIN AND IN THE ECH 2 MOUSE WE
SAW FATE SWITCH WHERE 1 WENT UP
AND 1 WENT DOWN.
IN THIS MOUSE WE SEE A DECREASE
OF BOTH CELL TYPES AND THIS IS
MORE STRIKING IN THE HIPPOCAMPUS
WHERE IF YOU LOOK IN THE
WILD-TYPE, THERE'S LOTS OF
TOMATO CELLS HERE AND YOU SLEEP
APNEA AND OBESITYY THE HUGE
POCKET WHERE IS THERE'S FEW
INTERNEURONS IN THESE MICE AND
WE QUANTIFIED ALL THAT AS WELL.
I WANT TO SHOW YOU A VIDEO OF
THESE MICE, SO THIS IS A KNOCK
OUT MOUSE HERE, THESE MICE HAVE
SPONTANEOUS EPILEPSY, YOU SEE
THESE PRONT PAWS HAVING THIS
BACK AND FORTH MOVEMENT AND THEN
KIND OF STARTS TO ARE HAVE THESE
RAPID MOVEMENTS, THIS IS UPDATER
GOING A SPONTANEOUS SEIZURE,
HERE'S ANOTHER EXAMPLE, THIS 1'S
KIND OF SEVERE, KIND OF A
CATATONIC SEIZURE, IT WILL
FREEZE UP AND FALL OVER, SO
THESE HOMOZYGOUS MOUSE ARE VERY
SICK IN TERMS OF THE FACT THEY
DISPLAY SPONTANEOUS DISEASURES
AND AGAIN THIS CORRELATES WITH
FEWER INTERNEURONS, SO YOU WE
CAN DO TESTS TO LOOK AT SEVERITY
OF THE SEIZURE, IT INDUCES
SEIZURES AND YOU SEE THAT THE
HOMOZYGOUS HERE IN RED, HAVE A
MUCH MORE SUSCEPTIBLE TO THIS
TASK, THEY UPDATER GO SEIZURES
OF MUCH MORE SEVERE LEVEL
COMPARED TO THE BLUE IN THE
HETEROZYGOUS WILD-TYPE MICE BE
AND WE'VE ALSO DONE
CERTAINLY--CERTAINLY LECT ROUGH
ATOM PHYSIOLOGY ON THESE, ON THE
LEFT THERE ARE 3 DIFFERENT
SUBTYPES AND YOU CAN SEE THE RED
DOTS AND GREEN DOTS AND BLUE
DOTS ARE FAIRLY WELL CLUSTERED
TOGETHER IF YOU LOOK IN THE
MUTANT THERE MUCH MORE SPARSE,
SO THIS INDICATES THE ACTUAL
PHYSIOLOGICAL CELLS IN THE
MUTANT THERE SEEM TO BE MUCH
MORE LIKE PROPERTIES OF MATURE
INTERNEURONS, BUT NOT ONLY DO
YOU HAVE FEWER INTERNEURONS BUT
THEY'RE ALL ELECTROPHYSIOLOGICAL
PROPERTIES ARE COMPROMISED AND
MAKE MORE AN IMMATURE STATE
RATHER THAN A MATURE
INTERNEURON.
WE HAD A LOT OF BEHAVIOR ASSAYS
ON THIS, I WILL TOUCH ON A
COUPLE.
THIS IS I FIELD TEST AND YOU PUT
THE MICE IN THE BIG 4 BY 4
BASICALLY PLEXIGLOSS BOX AND AT
THE CAMERA YOU CAN MONITOR
MOVEMENT AND QUANTIFY IT, THIS
IS A WILD-TYPE MICE, THEY KIND
OF PERFORM THE MITSD BUT
THEY'RE HAPPY TO EXPLORE A
ROUND.
THIS HOMOZYGOUS MICE, YOU CAN
SEE THEY AVOID THE MITSD, THEY
YOU CAN SEE IT'S A SIGN OF
ANXIETY, THEY DON'T WANT TO GO
IN MIDDLE, THEY WANT TO HUG THE
EDGE WHERE THEY FEEL SAFER AND
MORE SECURE, NOT ONLY DO THEY
SPEND TIME IN THE CENTER, BUT IN
PARTICULAR THE FEMALES MOVE A
LOT MORE, THEY TRAVEL A GREATER
DISTANCE AND ALSO A LOT FASTER.
SO 1 WAY TO THINK OF THIS IS IN
PARTICULARLY THE FEMALE MICE ARE
ZOOMING AROUND THIS BOX WHICH IS
A SIGN OF HIGH ANXIETY.
ANOTHER WAY TO LOOK AT IT, TO
TEST ANXIETY IS TO PUT MICE IN
THIS LIGHT DARK BOX, WITH KIND
OF A 6 C DECIBEL MICE WILL
PREFER IT IN THE DARK IN
GENERAL, BUT IF YOU SEE THESE
PREFERENCES OF THE LIGHT
CONTROLLER BOX
COMPARED TO THE DARK BOX.
WE HAVE DATA ON SOCIAL
INTERACTION, SOUNDS, MEMORY
DEFICITS, AND SEE THE FEMALES
HAVE MORE DEFICITS THAN THE MALE
MICE AND I WON'T GO INTO TOO
MUCH MORE ABOUT THAT.
NOW, AGAIN WE WANTED TO
PERFORM MULTIOHM ANALYSIS IN
THIS CASE BECAUSE THE MUTATION
IS DOING THINGS WE THINK ARE
CRITICAL IN BOTH THE
INTERNEURONS AND THE HYPOTHAM
MUSAND WE HOUSTON AND RICE
FOOTBALLESTED EMBRYONIC MGE AND
HEM BRIANAONIC HYPOTHALAMUS AND
AS WELL ADULT HYPOTHALAMUS AND
ADULT INTERNEURONS IN THE CORTEX
AND THIS IS HOW THE MAP LOOKS.
WE CAN TAKE OUT THE DIFFERENT
SUBTYPES AND DO ALL KINDS OF
ANALYSIS AND EMBRYONIC 1S WE
SEE, INDICATORS OF ALL THESE
HYPOTHALAMIC NUCLEI THAT EXIST,
WE SEE THE 2 TYPES OF
INTERNEURONS AND THEN THE
HYPOTHALAMUS AND I WILL
HIGHLIGHT 2 THINGS WE'VE LEARNED
FROM THIS DATA THAT'S
INTERESTING, 1 IS THAT IF WE
FOCUSED ON P60 CORTEX SO THESE
ARE THE MATURE INTERNEURONS WE
CAN AGAIN LOOK AT DEFERENTIALLY
EXPRESSED GENES, WE CAN LOOK AT
THE DIFFERENT TERMINOLOGY
FACTORS THAT I HAVE BUT WHAT'S
INTERESTING TO US IS IF YOU TAKE
THESE MOST DIFFERENTLY EXPRESSED
GENES HERE AND MAP THEM ON TO
VARIOUS HUMAN DISEASES, WHAT YOU
SEE IS A LOT OF HITS ON
EPILEPSY, EPILECTIC SYNDROME,
THIS IS INTERESTING BECAUSE A
LOT OF THE YEENS THAT ARE MOST
DIFFERENTLY EXPRESSED IN THESE
MUTANT MICE ARE ASSOCIATED WITH
EPILEPSY WHICH WE SEE THESE MICE
HAVE, THAT'S 1 WAY WE CAN USE
THIS DATA TO KIND OF CORRELATE
CHANGES IN GENE EXPRESSION WITH
DISEASE.
IF WE LOOK IN THE ADULT
HYPOTHALAMUS AND WE BREAK IT
DOWN IN BETWEEN CELLS THAT COME
FROM THE WILD-TYPE AND
HOMOZYGOUS MICE SO WE IDENTIFY
NEURONS, OLIGIOS ASTRO SIGHTS
AND 10 SAITS AND YOU CAN SEE A
DIFFERENCE HERE IN THE ASTRO
SLIGHT CLUSTER WHERE THE MUTANT
AND WILD-TYPE MICE ARE CLUSTERED
DIFFERENTLY AND WE CAN AGAIN
EXTRACT SPECIFICALLY THESE
SUBSETS AND KIND OF LOOK AT THE
DIFREBTIAL GENE EXPRESSION AND
THAT THERE'S A WHOLE POPULATION
OF ASTRO SIGHTS THAT ARE
BASICALLY MISSING IN THE
HOMOZYGOUS MICE IN TERMS OF GENE
EXPRESSION AND FUNCTION.
ANOTHER THING THAT WAS
INTERESTING IS THAT IF YOU LOOK
AT THE PROPORTION OF CELLS,
AGAIN, ASTRO SIGHTS ARE BLUE
HERE, BASED ON THIS, IT LOOKS
LIKE THERE'S MORE ASTRO SIGHTS
IN THE MUTANT COMPARED TO
WILD-TYPE.
IS THAT TRUE?
IT IS.
WHEN WE UMPIRESUNE O STAINING
FOR THIS MARKER OF ASTRO SIGHTS
WE SEE A LOT MORE ASTRO SIGHTS
IN THE MUTANT COMPARED TO THE
WILD-TYPE, SO IT'S NOT SOMETHING
WE COULD HAVE PREDICTED, NOT
SOMETHING WE HAD AN IDEA ABOUT,
BUT IT LOOKS LIKE THIS MUTATION
CAUSES SIGNIFICANT INCREASE IN
ASTRO SIGHTS AND PABLLY A LOSS
OF A PARTICULAR SUBSET OF ASTRO
CITES IN THE WILD-TYPE.
NOW TAM, IS A REGION WHERE CURES
CAN ENTER THE BLOOD STREAM AND
HAVE CROSS TALK IN GENERAL IN
THE REST OF BRAIN, THERE'S
SIGNIFICANT RESTRICTION FOR
ANYTHING TO CROSS THE BLOOD
BRAIN BARRIER BUT THIS REGION IN
PARTICULAR, THESE TAN O SIGHTS
ARE CRITICAL WHERE THE BRAIN CAN
COMMUNICATE WITH THE BLOOD
STREAM AND THEY HAVE A VARIETY
OF FUNCTION IN AND ROLES AND
VARIETY OF FUNCTIONS WE THINK
ARE AFFECTED IN THESE MICE, SO
WE TOOK CROSS SECTIONS TO
AMENOSTAIN THESE REGIONS WITH
GFAP, SO THE ASTROCYTES MARKER
AND THESE ORGANIZATION IN TERMS
OF THE CELLS, IF YOU LOOK IN THE
MUTANT MOUSE WHICH YOU SEE A
STRIKING DISRUPTION OF THIS
ORGANIZE EGG, THERE'S WAY MORE
NK CELLS LIAISONS RUPTED MANNER
HERE, ASTROCYTES ARE ON THE
EDGE, SWITCHED AND NOT IN THE
MIDDLE AND YOU CAN SEE STRIKING
DIFFERENTS IN THESE TANYCYTES
MORPHOLOGY.
SO THERE'S SOMETHING GOING ON
THAT'S PERTURBING THESE CELLS,
BETWEEN THE BLOOD AND HORMONES
IN THE BRAIN AND THIS ALL FITS
INTO WHY THESE MICE POSSIBLY
HAVE OBESITY AND PERTURBATION OF
BEHAVIORIAL DEFICITS AND FEEDING
DEFICITS.
OKAY, SO, I WILL RUN THIS LAST
PART VERY QUICKLY.
SO THE LAST 2 THINGS AGAIN WE'RE
KIND OF TAKING A SLEDGE HAMMER
APPROACH SO NOW WE WANT TO LOOK
AT GENES THAT ARE REALLY MORE
AFFECTED, MORE CORRELATED WITH
HUMAN DEC AND 1 IS CALLED THIS
GENE ARC'S TRANSCRIPTION FACTOR.
THIS HAS BEEN WELL STUDIED IN
THE LITERATURE, ON THE X
ROAMSOME AND IS RESPONSIBLE FOR
ABOUT 10% OF X-LINKED
INTELLECTUAL DISABILITY
DISEASES.
YOU CAN SEE THERE'S A TON OF
POINT MUTATIONS IN THIS GENE,
AND MORE RECENTLY SINCE AT
X-LINKED IT WAS THOUGHT TO
PRIMARILY OR ONLY AFFECT MAILS
BUT THERE'S RECENT EVIDENCE NOW
THAT FEMALE CARRIERS, HETZ HAVE
MILD AND SEVERE PHENOTYPES.
WHAT'S INTERESTING FOR US IS
THIS GENE IS KNOWN TO BE
EXPRESSED IN THE GANGLIONIC
EMINENCES AND STUDY VS KNOCKED
OUT THIS YEEN IN POST MITOTIC
CELLS BUT THERE HASN'T BEEN MUCH
STUDIED WHEN YOU IMK IT OUT IN
CYCLING CELLS.
SO WE USE OUR NKX2.1 CRE TO SEE
HOW IT AFFECTS THE CELLS FATE
AND ALLOWS US TO LOOK AT HOW
ETICSERY ZYGOUS MOUSE AFFECTS A
FEMALE BECAUSE THAT HASN'T
REALLY BEEN TODAYED.
SO JUST TO RUN THROUGH THIS DATA
PRETTY QUICK, THE MALES ARE VERY
SICK, THEY'RE UNDERWEIGHT, THEY
ALL DIE BY 8 WEEKS.
WHERE PHENOTYPICALLY THE FEMALES
HESES ARE NORMAL SIZE, THE MALES
ARE KNOCKED OUT, THE FEMALES
AGAIN BECAUSE IT'S X-LINKED.
AND HERE'S VIDEOS, SO THE MALES
ARE VERY SICK, HERE'S INFANNITE
SEIZURES, SPASMS YOU SEE EVEN
WHEN THE PUPS ARE ABOUT 8 WEEKS
OLD, THIS SHOULD LOOK FAMILIAR
FROM THE K4 MMOUSE AND THESE
MICE ARE ALSO HYPEY ACTIVE.
WE PUT THEM THROUGH THE SAME
SEIZURE SCORE, AGAIN ONLY
FEMALES BECAUSE THE MAILS DIE
INJECTING THE DRUG CAUSES THEM
TO DIE, BUT WE SEE THE FEMALES
HAVE A HIGHER SUSCEPTIBILITY
SEIZURES AND AGAIN WE'RE
CURRENTLY PUTTING THEM THROUGH A
BATTERY OF ASSAYS.
WHAT YOU CAN SEE IS REALLY
STRIKING LOSS OF INTERNEURONS SO
THIS IS THE CORTEX HIPPOCAMPUS,
THIS IS THE MORMAL AMOUNT OF MG
DRIVING THE NEURONS.
THE FEMALES AGAIN HAVE DECREASE
AND THE MALE KNOCK OUTS HAVE A
STRIKING LOSS OF INTERNEURONS
AND THAT CAN BE SEEN HERE AND
AGAIN QUANTIFIED WHERE YOU HAVE
JUST A STRIKING LOSS, 80-90%
LOSS OF INTERNEURONS IN THIS
MOUSE.
THIS IS MORE SEVERE THAN THE
OTHER STUDIES THAT KNOCKED OUT
ARX POST MITOTICALLY, THEY
DIDN'T HAVE NEARLY THIS AMOUNT
OF CELL LOSS OF 1 COOL THING
WE'RE TRYING TO INDGREAT INTO
THE LAB, IS CELL
TRANSCRIPTOMICS, BUT WITH
SPATIAL TRANSKRPT ORDER OF
MICRONSICS YOU CAN LOOK AT
SPATIAL INFORMATION IN DIFFERENT
YALE GENE EXPRESSION AS WELL AS
LOOK AT NONCELL AUTONOMOUS
EFFECTS.
SO THIS IS JUST KIND OF INITIAL
DATA WE'VE HAD FOR A COUPLE
WEEKS, BEING LOAMACYING AT THE
WILD-TYPE IN THE HETEROZYGOUS
MOUSE WHERE WHAT YOU'RE DOING IS
RATHER THAN HAVING A SINGLE CELL
REAS LIEWGZ, LOOK AT A CELL
REGION AND LOOK AT GENE
EXPRESSION BOTH IN TERPS OF THE
SPATIAL MAP, AS WELL AS IN A
UMAP PLOT.
YOU CAN LOOK AT GENE EXPRESSION
PROFILE.
SO AGAIN, HERE'S THE WILD-TYPE
AND THEN IN THIS CASE, THE HAT
FEMALE, CAN YOU SEE THIS IN THE
MALE, BUT YOU CAN SEE STRONG
REGULATION OF P A LB-ALBUT MIN,
AND WE'RE TRYING TO SINGLE CELL
DATA AND TRANSCRIPTIONAL DAT TO
HAVE A COMREHENSIVE WAI TO LOOK
AT SPATIAL INFORMATION AND CELL
AUTONOMOUS EFFECTS AND CELL
NONAUTONOMOUS EFFECTS LIKE THIS.
OKAY, SO JUST TO SUMMARIZE, I'VE
SHOWED YOU HOW WE DEVELOPED A
EPIGENOME AT LIAISONS WITH A
MULTIMODAL APPROACH TO
CHARACTERIZE THE CELLS IN THE
EMBRYONIC BRAIN AS THEY'RE
MAKING CRITICAL DECISIONS IN
CELL FATE.
I'VE SHOWN 2 APPROACHES WE USE
TO TAKE A SLEDGE HAMMER TO YEEN
REPRESSION AND ACTIVATION AND
SHOW HOW THAT AFFECTS BOTH THE
FATE AND THE PERCENTAGES OF
CELLS AND CELL LOSS.
IN SPL CASES REALLY STRIKING
SEVERE MITRAL SITS IN ANIMAL
BEHAVIOR AND NOW WE'RE WELL
POSITIONED TO KIND OF LOOK MORE
SPECIFICALLY AT YEENS WE KNOW
ARE CRITICAL BOTH IN INTERNEURON
DEVELOPMENT AS WELL AS HUMAN
DISEASE, AND KIND OF TAKE THIS
PIPELINE TO REALLY HAMMER HOME
AND HOPEFULLY PROVIDE IMPORTANT
INSIGHTS INTO SOME OF THESE
DISEASES.
SO I HOPE YOU KIND OF CONVINCED
YOU HOW WE'RE TRYING TO USE A
VARIETY OF APPROACHES TO REALLY
UNDERSTAND HOW YOU GO FROM THESE
KIND OF HETEROGENEOUS POPULATION
IN THE EMBRYO TO THIS INCREDIBLE
DIVERSITY IN THE ADULT BRAIN,
HOW GENETIC FACTORS AND
EPIGENETIC AND GENE REGULATORY
FACTORS COMBINE WITH THE BRAIN
ENVIRONMENT TO INFLUENCE FATE
AND MATURATION OF THESE CELLS
BOTH IN NORMAL DEVELOPMENT AND
MOST CRITICALLY, WHAT GOES WRONG
IN DISEASE VARIOUS DISEASES.
SO I JUST WANT TO MAKE EVERYBODY
IN MY LAB IN PARTICULAR, 3 POST
DOCS HIGHLIGHTED HERE IN THE
VARIOUS PROJECTS I TALKED ABOUT.
A VARIETY OF POST BACKS,
IMPORTANT COLLABORATOR,
PARTICULARLY PEDRO ROCCA, THE
Mc BRAIN LAB FOR
[INDISCERNIBLE] LAB, AND RYAN
[INDISCERNIBLE] RUNS THE
BIO-INFORMATICS CORE THAT WE
COULDN'T DO A LOT WITHOUT.
THAT'S IT.
HAPPY TO TAKE QUESTIONS.
>> ANY QUESTIONS?
>> CAN I ASK A QUESTION ABOUT
YOUR MARK ON DISRUPTION OF H3 K4
TRI-METHYLATION?
DID YOU DO IT SPECIFICALLY FOR
THE NK2.1 LOCUST OR IT WAS DONE
ACROSS THE SPECTRUM OF ALL
GENES?
>> IT'S A FLOCKS ALLELE, SO WE
USE NKX2.1 CRE, SO THE MUTATED
ALLELE WAS ONLY EXPRESSED IN
THOSE REGIONS AND WE DID CONFIRM
THAT, THE MUTANT ALLELE IS
HA-TAG SO WE CONFIRMED BOTH
REDUCTION IN THE K4 METHYLATION
-- IN THE MG AND HYPOTHALAMUS
AND ALSO CONFIRMED UPREGULATION
OF HA SPECIFICALLY IN THOSE
REGIONS.
SO MY PREDECLARATION IS IF WE
DID THIS GLOBALLY AND THE PEOPLE
THAT MADE THE MOUSE DID IT,
THESE ARE DIFFERENT CRE DRIVER
BUT THE MICE DIED EARLY, SO I
THINK THE WILD-TYPE ALLELE IS
INTACT BUT I THINK UPREGULATION
OF THIS MUTANT ALLELE, ACROSS
THE BOARD, LIKE WITH A PAN CRE
DRIVER WOULD ALMOST CERTAINLY
RESULT IN ANIMAL DEATH WHICH THE
GROUP THAT DEVELOPED THIS MOUSE
LINE SAW USING A DIFFERENT CRE
DRIVER.
>> AND I ASSUME THAT BY DOING
THAT, YOU EFFECTIVELY DOWN
REGULATE THE ACTIVITY OF ALL THE
ENHANCERS OF NKX2.1 AND IF YOU
WOULD LIKE TO GO INTO THE
GRANULARITY OF THE REGULATORY
PROGRAM, YOU CAN PROBABLY
SOMEHOW GO 1 BY 1 AND SOMETHING
LIKE THAT AND SEE HOW YOU WERE
TALKING ABOUT THIS EVOLUTIONARY
WAYS OF STABILIZING GENE
EXPRESSION SO THAT PROBLEM IS
THE [INDISCERNIBLE] ENHANCERS
GENETIC ACTIVITY OF MULTIPLE
SHOULD KNOWN TO DECIPHER THAT
INSIDE OF NKX2.1 LOCUST, I'M
GUESSING LIKE CRSPR WOULD BE
NEEDED TO BREAK THE PIECES?
>> YEAH, THE LAST PIECE OF DATA
ON THAT STORY IS CUT AND TAG
WITH THE K4 TRI-METHYLATION, SO
THAT WE CAN LOOK AT DIFFERENCES
THROUGHOUT THE GENOME.
BECAUSE AGAIN, IF IT'S NKX2.1
WERE MORE RESIST ABT AS YOU WERE
HINTING AT, MAYBE THE 2.1 LOCUST
WILL SHOW GREATER RESISTANCE TO
THE PROTURBATION THAN OTHER LOCI
SO THAT'S THE LAST PIECE OF DATA
THAT CAN SPEAK TO THAT THAT
WE'RE KIND OF WAITING ON RIGHT
NOW.
BUT THERE ARE OTHER WAYS YOU CAN
MANIPULATE THAT WITH CRSPR, AND
OTHER WAYS TO LOOK AT THAT
EFFECT.
IT'S TRICKY, TOO, BECAUSE 50% OF
THESE HOMOZYGOUS MICE DIE, SO
WE'RE LOOKING AT THE HEALTHIEST
OF THE SICK MICE THAT SURVIVE
AND BECAUSE THEORETICALLY IT'S A
50/50 RATIO OF WHETHER THE
MUTANT ARK LEGAL IS THERE, THE
WILD-TYPE ALLELE IS THERE, IT
MIGHT NOT BE 50/50, WE HAVEN'T
-- IT'S VERY COMPLICATED TO LOOK
AT THAT, BUT IT'S A DIRTY MOUSE
IN THE SENSE THAT THERE'S GOING
TO BE VARIABILITY JUST
THEORETICALLY AND WHICH ALLELE
IS INTEGRATED INTO WHICH SITE
AND SOME SITES MIGHT HAVE THE
MUTANT ALLELE AND THOSE SITES
MIGHT BE MORE CRITICAL FOR
PARTICULAR GENE ACTIVATION AND
THUS GIVE A HIGHER PHENOTYPE.
SO, MOUSE MODELS REALLY NICE FOR
FOR THINGS BUT IT IS SORT OF
DIRTY IN A SENSE OF HAVING THAT
LEVEL OF GRANULARITY.
>> [INDISCERNIBLE]
>> WE DON'T HAVE ANYMORE
QUESTIONS ONLINE AND I WOULD
LIKE -- 1 MORE?
>> I WAS WONDERING, YOU KNOW I
MAY HAVE MISSED SOME OF YOUR
OBSERVATIONS BUT YOU SAID YOU DO
A KNOCK OUT IN THE EXPERIMENT IN
WHICH YOU DO THE KNOCK OUT OF
THE ENHANCER OF THE STCH2.
IT SEEMS LIKE AT THE END YOU GET
A DIFFERENT PROPORTION OF THE 3
TYPES OF 3 TYPES OF CELLS AND I
WAS WONDERING WHERE IS THIS
AFFECT COMING FROM?
IS IT COMING FROM A CHANGE IN
FATE OF YOUR CELLS?
IS THIS COMING FROM A DIFFERENT
CELL MIGRATION AND
DIFFERENTIATION?
IT COMING FROM CELL DIVISION,
THE CHANGE OR PROPORTION OF
THESE CELLS?
SORE IS THIS COMING FROM CELL
DEATH THAT AFFECTED, YOU KNOW,
THESE CELLS DIFFERENTIALLY SO
THAT YOU END UP WITH THE
DIFFERENTIAL --
>> THAT'S AN EXCELLENT QUESTION
AND YOU HIT ON ALMOST ALL THE
POSSIBILITIES, I THINK.
SO WE HAVEN'T NAILED THAT DOWN.
I WILL SAY WE LOOK AT CELL
DEATH, WE DON'T SEE SIGNIFICANT
CHANGES.
ONE NUANCE AND DEVELOPMENT OF
THE PBS ST CELLS THAT I
MENTIONED IS THAT THESE CELLS
START TO BE BORN AROUND E11ISH
AND THEY'RE BASICALLY CONTINUED
TO BE BORN UNTIL ABOUT THE
ANIMAL'S -- UNTIL THE ANIMAL IS
BORN AT ABOUT E18.
SO YOU HAVE A RANGE OF 7 DAYS
THAT'S PRODUCING THESE
INTERNEURONS NOW THE PB AND SST
CELLS ARE NOT PRODUCED OVER THAT
TIME, SO IN THE BEGINNING
THERE'S A BURST OF SOPHISTICATED
MATOSTATIN CELLS AND THEN THE
PRODUCTION GOES DOWN OVER TIME
ON DURING EMBRYO GENESIS,
[INDISCERNIBLE] CELLS ARE MORE
CONSTANT.
SO IF YOU LOOK AT E12, E13,URE
MAKING A 50/50 MIX OF PB AND SST
CELLS BEING BORN, IF YOU LOOK AT
E16, IT'S AN 80/20 MIX OF
[INDISCERNIBLE] CELLS OVER
SOPHISTICATED MAT O CELLS.
SO IF YOU HAVE A DEVELOPMENT OF
MUTATION LIKE I THINK IS
HAPPENING IN THE ECH 2, WHERE
YOU ARE KICKING CELLS OUT OF THE
CELL CYCLE TOO EARLY, WHAT WILL
HAPPEN IF YOU'RE MAKING TOO MANY
TOO EARLY, THERE'S A BIAS FOR
THOSE TO BE SOPHISTICATED
MATOSTATIN CELLS AND IF YOU'RE
DEPLETING THE PROGENITOR POOL
LATER ON SO YOU HAVE FEWER CELLS
BEING BORN LATER ON WHERE PB
CELLS DOMINATE, YOU WILL HAVE
FEWER PB CELLS.
SO WE LOOK AT THAT A LITTLE BIT.
THE DATA WASN'T AS CONVINCING AS
I HOPED BUT MY BEST GUESS
BECAUSE A LITTLE BIT OF WHAT WE
KNOW ABOUT ECH2 AND 3 INNESS
KACCTER IS THAT TOO MANY CELLS
ARE BORN TOO EARLY, DEPLETING
THE PROIENITTOR POOL AND BECAUSE
OF THE TIME COURSE OF
SOPHISTICATED MAT O 7 PB-CELLS
ARISE, THAT EXPLANATION OF AND
OF ITSELF IS SUFFICIENT TO HAVE
A DECREASE OVERALL IN THE NUMBER
OF INTERNEURONS THAT WE SEE AND
ALSO HAVE THIS FATE SWITCH.
SO MAYBE, YOU KNOW FATE SWITCH
IS A DANGEROUS WORD.
I THINK WHAT'S MORE ACCURATE IS
THAT INCREASED PRODUCTION OF
INTERNEURONS EARLY AT A TIME
WHEN YOU'RE BIASED FOR
SOPHISTICATED MATOSTATIN CELLS
AND DECREASE PRODUCTION LATER AT
A TIME WHEN YOU'RE BIASED FOR PB
CELLS, THAT'S HOW YOU GET THE
FATE SWITCH APPEARANCE BUT I
DON'T THINK THE INDIVIDUAL CELLS
ARE SWITCHING FATE ALTHOUGH WE
DON'T HAVE DIRECT EVIDENCE AGAIN
THAT I THINK THIS OTHER SCENARIO
IS MUCH MORE LIKELY FOR THE
EXPLANATION.
>> [INDISCERNIBLE].
>> OKAY.
>> [ APPLAUSE ]
>> THANK YOU.