>> WELL, IT'S MY PLEASURE TO INTRODUCE TODAY'S SPEAKER JOHN RUBENSTEIN, HE PRESENTLY HOLDS THE NINNA IRELAND DISTING WIRED PROFESSORSHIP IN CHILD PSYCHIATRY BUT HE'S ALSO A MEMBER OF THE BIOMEDICAL SCIENCES AND BIOMEDICAL SCIENCE PROGRAM AND THE MOLECULAR INSTITUTE OF MEDICINE IN SAN FRANCISCO. JOHN CLEARLY LIKES NORTHERN, CALIFORNIA, HE GOT HIS BS, Ph.D. AND MD FROM STANFORD. HE LEFT STANFORD AFTER HIS Ph.D. AND MD DEGREE AND WENT TO THE PASTEUR INSTITUTE WITH JOE COB AND CAME BACK TO STANFORD TO CONTINUE HIS INTERNSHIP, RESIDENCE SCHEFELLOWSHIP IN PSYCHIATRIC MEDICINE AT STANFORD,UT HIS POST DOC, I HAVEN'T TALKED TO JOHN ABOUT THIS BUT I BELIEVE HIS POST DOC HAD A BIG IMPACT ON HIM, IT'S ON HIS WAY TO STUDY NEURODEVELOPMENT WHICH HE CONTINUED TO STUDY IN ELEGANT WAYS OVER THE LAST 20 OR SO YEARS. HE IS OVER 200 PEER REVIEWED PAPER AND HE'S REALLY A FINE EXAMPLE OF SOMEONE WHO'S COMBINING TRANSLATIONAL AND BASIC APPROACHES TO STUDY NEURODEVELOPMENT WITH AN UNDERSTANDING TO GET AN UNDERSTANDING OF NEUROPSYCHIATRIC DISORDERS. I LEARNED HE HAS 3 RO-1S RIGHT NOW WHICH IS VERY IMPRESSIVE BUT I THINK PERHAPS MORE IMPRESSIVE AMONG JOHN, MANY AWARDS AND DISTINGUISHED ACCOLADES IS THE FACT THAT I BELIEVE HE'S HAD A CONSISTENT RO-1 SINCE 1992. THAT IS PRETTY PHENOMENAL. HE'S ON THE EDITORIAL BOARD OF MANY JOURNALS INCLUDING DEVELOPMENT, GENES AND NEURODISORDERS, AGAIN BRIDGING THE GAP BETWEEN BASIC AND TRANSLATIONAL. WHEN JOHN AND I WERE FIRST TALKING ABOUT HIM COMING HERE TO GIVE A TALK, HIS REQUIREMENT WAS THAT THE CHERRY BLOSSOMS BE OUT. [LAUGHTER] AND WHEN MARCH CAME AROUND AND IT WAS STILL SNOWING AND NOTHING WAS OUT THERE, I WAS GETTING A LITTLE WORRIED AND I E-MAILED HIM EVEN A WEEK AGO AND SAID, JOHN, IT'S NOT REALLY THAT WARM YET HERE, BUT OUR TIMING WAS REALLY PERFECT AND JOHN WAS NICE ENOUGH T BRING 2 DAYS OF BEAUTIFUL SOUTHERN CALIFORNIA, I DON'T THINK IT'S ACTUALLY NORTHERN CALIFORNIA WEATHER WITH HIM WHICH WE APPRECIATE. AND IN TALKING, OUR TIMING WAS ALSO GOOD BECAUSE WE D'T EXACTLY KNOW WHAT'S GOING TO HAPPEN NEXT WEEK WITH THE GOVERNMENT. SO THIS IS A GOOD TIME TO HAVE A LECTURE. SO I'M REALLY PLEASED THAT JOHN IS HERE TODAY AND HE'S GOING TO BE TALKING ABOUT TRANSCRIPTIONAL REGULATION, I'M READING THIS OFF OF YOUR TRANSCRIPTIONAL REGULATION OF 2 PHASES OF SONIC HEDGE HOG DEPRESSION DRIVE MGE DEVELOPMENT AND WE'RE HAPPY TO HAVE YOU, WELCOME. [ APPLAUSE ] >> HI, I'VE HEARD THIS IS A VERY TOUGH AUDIENCE SO JIM BADI HAS LED ME HIS HARD HAT. BUT I'LL TAKE IT OFF BECAUSE IT'S A BIT WARM BUT WHEN YOU ASK THE DIFFICULT QUESTIONS I MAY PUT IT BACK ON. [LAUGHTER] WELL, THANKS FOR COMING TO LIFIC TO ME, IT'S--LIFIC--LISTEN TO IT'S GREAT TO COME TO NIH WHERE THERE ARE SO MANY YOUNG FRIENDS OF MINE TO SEE AGAIN AND REMINISCE. WHAT I WILL TELL YOU ABOUT TODAY IS A STUDY HAVE BEEN ONGOING FOR ABOUT 10 YEARS IN COLLABORATION WITHINER WEST FALL AND YOUNG GUN SAIL AND THEY'VE BEEN WONDERFUL AND WE'VE HAD SYNERGISTIC AND WE'VE GOTTEN THINGS DONE THAT WOULD HAVE BEEN IMPOSSIBLE. THIS IS A PICTURE FROM YESTERDAY. THIS IS THE HOUSE, LOOKING DOWN AT YOUR BEAUTIFUL CITY. BUT UNFORTUNATELY THE SUBJECT FOR TODAY'S TOPIC IS SOMETHING ELSE, IT'S THE DEVELOPMENT OF THE TELENCEPHALON FOCUS TED ALMOST ENTIRE O ON THE MOUSE. WE THINK THE MOUSE IS PRETTY MUCH THE SAME AS THE HUMAN. THIS IS A 1 HEMISPHERE OF A TELENCEPHALON SHOWING THE CORTEX AND THE BASAL GANGLIA AND THE PREOPTIC AREA AND HERE I COLORED THE ZONE OR THE STEM CELLS FOR THESE STRUCTURES IN DIFFERENT COLORS, GREEN FOR THE CORTEX, BLUE FOR THE BASAL GANG RIA AND RED FOR THE PREOPTIC AREA TO INDICATE THAT THE PROGENITORS IN EACH OF THESE REGIONS ARE EXTREMELY DIFFERENT AND HAVE ALREADY PROGRAMMED THE ABILITY TO MAKE DIFFERENT KINDS OF NEUROTRANSMITTERS SO THE CORTICALEf PROGERATEDI TTORS AS FAR AS WE KNOW IN MOUSE MAY ALMOST EXCLUSIVELY MAKE GLUTEA MATE FOR THE PROGENITORS, WHERE THEY'RE PROGRAMMED TO MAKE GABBA AND THE PREOPTIC AREAS MORE COMPLICATED BUT THEY MAKE FETAL CO LINE. SO THIS IS NOT THAT DIFFERENT THAN THE SPINAL CORD HAVE YOU HAVE A SIMILAR SEGGREGATION OF GLUTEA MATE GOBBA AT THE MIDDLE AND FETAL CO-LINE AT THE BOTTOM. IF YOU'RE A PROJECTION NEURON COMING OUT OF THE STEM CELLS YOUR TYPICAL PROGRAM IS TO MIGRATE RADIALLY AWAY FROMnxE THE ZONE AND THAT'S WHAT THESE ARE MEANT TO INDICATE. RADIAL MIGRATION AWAY FROM THE ZONE, WHERE THE PROJECTION SETTLED IN NUCLEI OF THE BASAL GANGLIA AND CONTAIN INDEED GLOBEUS PALATUS OR THE CEREBRAL LAYERS IN HYPOCAMPUS. >> ONE OF THE STRUCTURES TODAY IS THE GLOBEUS PALAT US, IT'S 1 OF THE LARGER OF THE BASAL GANGLIA AND IT COMES FROM THIS PROGENITOR ZONE CALLED THE MGE OR MEDIAL GANGLIONIC EVIDENCE, I'LL REFER TO IT AS MGE FOR THE REST OF THE TALK. SURPRISINGLY WHEN WE FIRST FOUND THIS ABOUT 15 YEARS AGO, THE THE MGE ALSO SEEMS TO MAKE OVER HALF OF THE INTERNEURONS THAT POPULATE THE CEREBRAL CORTEX AND HIPPOCAMPUS AND THAT'S WHAT THESE ARE MEANT TO INDICATE, THAT THERE IS A TANGENTIAL MIGRATION OF THE INTERNEURONS FROM THE BASIAN DECODER ALGANGLIA ZONES TO THE OVERLYING CORTEX AND HIPPOCAMPUS. LIKE WISE THERE'S A SIMILAR TANGENTIAL MUSEUMIGRATION THAT BRING ITS INTO THE CAN YOU TANMENT OF THE OTHER REGIONS OF THE MGE, POA. SO THERE SEEMS TO BE AN OLD EVOLUTIONARY PROGRAM WHEREBY DIFFERENT CELL TYPES ARE PRODUCED IN THESE DIFFERENT DORSAL SUBDIVISIONS, THE TELENCEPHALON AND THEN TANGENTIAL MIGRATIONS IN CELL TYPES THAT WOULDN'T BE MADE IN A PARTICULAR REGION TO ARRIVE TO THE FINAL DESTINATION. AND AS FAR AS HOST OF YOU KNOW IN THE CEREBRAL CORTEX, A TYPICAL YELLOW PATHWAY GIVES RAMTAL CELL, THESE ARE THE EXCITATORY PROJECTIONS, ABOUT 80% OF THE NEURONS ARE THIS CELL TYPE, THEIR ACTIVITY IS REGULATED BY THE INTERNEURONS, THE GABAergic LOCAL CIRCUIT NEURONS AND YOU CAN SEE THIS PICTURE INDICATES MANY DIFFERENT KINDS OF LOCAL CIRCUIT NEURONS SOME THAT INNOVATE THE DENDRITES OF THE PATHWAY GIVES RAMTAL CELLS, SOME TO INNERIVATE THE CELL BODY AND SOME TO INTERIVATE THEAXON AND THEREBY ENABLING THEM TO CONTROL PERCEPTION OF INFORMATION, PROCESSING INFORMATION OR EXITING OF INFORMATION FROM THE EXCITATORY NEURONS. AND MANY OF YOU ALSO KNOW THERE'S A LOT OF--I WOULDN'T SAY EXCITEMENT BUT INTEREST IN THE POSSIBILITY THAT PSYCHIATRIC DISEASES SUCH AS SCHIZOPHRENIA ARE DUE TO DEFECTS IN INTERNEURON DEVELOPMENT AND FUNCTION. SO THERE'S CURRENTLY AT LEAST A LOT OFRç: EMPHASIS ON HAPPENING THE DEVELOPMENTAL PROGRAMS FOR GENERATION OF LOCAL CIRCUIT NEURONS FOR THE SEREBERAL CORTEX, VIS A VIS NEUROPSYCHIATRIC ILLNESSES. A HIGHER RESOLUTION MAPS OF SUBDISCIPLINARY DITIONS OF THE BASAL GANGLIA PROGENITOR ZONES HAVE BEEN ESTABLISHMENTED. THIS IS A COLLABORATOR OS COR, PRODUCED AND IT IS PUBLISHED AND HERE WE HAVE 4 PLANES OF SECTION OF THE EMBRYONIC TELENCEPHALON, ROST ILLEGALS ON YOUR LEFT AND CODDLE ON YOUR RIDE. THE MEDE YALE GANGLIONIC EVIDENCE IS SHOWN IN RED AND THE STRUCTURE JUST DORSAL TO IT, CALLED THE LATERAL OR LGE IS TO THE LEFT AND YOU YOU MAY BE ABLE TO SEE THAT WE INDICATED THE EXISTENCE POTENTIALLY OF UP TO 4 DIFFERENT SUBDIVISIONS, I'M SORRY 5 DIFFERENT SUB DIVISIONS WITHIN THE STEMÎe CELLS OF THESE STRUCTURES AND PART OF MY TALK TODAY WILL BE ABOUT WHAT THE DIFFERENT SUBDIVISIONS MAY BE DOING, BUT THIS IS STILL WORK IN BRING AND IT'S A MODEL, IT'S NOT MEANT TO BE THAT THERE ARE THESE CARDINAL SUBDIVISIONS OF THE BASAL GANGLIA. THE STARS OF THE TALK ARE 4 TRANSCRIPTION FACTORS. NKX2.1 NAMED BY MARSHAL NUREMBERG, HE'S AT NIH. THE THOSE WERE THE NK GENES, NUREMBERG AND CHEM GENES THEY DISCOVERED IN INVERTEBRATES. THIS IS A MOUSE HOM LOG OF THAT AND THIS GENE IS A TRANSCRIPTION FACTOR IN THE EMBRYONICKIA AND EXPRESSION DOMAIN CAN AND THEN THE LHX, GENES, 6 AND 8, THESE ARE DISCOVERED IN SEVERAL LABS INCLUDINGINER WEST FALL'S LAB AND THERE ARE LIMB HOMEIO BOX TRANSCRIPTION FACTORS THAT HAVE EXPRESSION PATTERN TO NK2.1 AND THEY CHECK THE EXPRESSION,oVT ALMOST IDENTICAL, PROBABLY ALL THE CELLS IN THE PROGENITOR ZONE EXPRESS THESE 3 TRANSCRIPTION FACTORS. AND THEN A FOURTH TRANSCRIPTION FACTOR IS TLX FAMILY, TLX IS THE EARLIEST EXPRESSION MEMBER OF THAT FAMILY AND IT IS EXPRESSED LIKE THE OTHER 3 MET MEDIAL GANGLIONIC EVIDENCE AND YOU CAN SEE IT'S EXPRESSED IN THE LATERAL OR GANGLIONIC, OH LGE, AND I WILL NOT BE TALKING ABOUT THAT MUCH TODAY EXCEPT THAT IT IS THE CENTERPIECE OF 1 OF MY GRANTS. SO TOM IS HERE AND UNFORTUNATELY I DON'T HAVE TIME TO TALK ABOUT THAT WORK. SO I'LL START BY REVIEWING PUBLISHED INFORMATION ABOUT THE FUNCTION OF OF LH NK2.1 AND LH6 AND 8, AS DISCOVERED BY ATHAT WILL SIS OF MOUSE--ANALYSIS OF MOUSE MUTANTS. THE FIRST MOUSE MUTE ANT I'LL TALK ABOUT NKX2 WENT 1 WHICH WAS MADE BY SHIRR O KINEMURA AT NIH. OKAY, SO THIS IS THE EXPRESSION OF NK2.1 IN A WHILED TYPE--WILD-TYPE MOUSE IN THE THE AREA. DESPITE THE LACK OF PROTEIN THE GENE IS EXPRESSED, THE RNA IS STILL PRESENT ALTHOUGH THE AMOUNT OF REGION THAT EXPRESSES IT IS REDUCED. AND HERE'S EXPRESSION OF LHX 8 AND 6. AGAIN, VERY SIMILAR EXPRESSION PATTERN TO NK2.1 AND AS CAN YOU SEE IN THE NK2.1 MILL MUTE ANT NEITHER GENE IS EXPRESSED SO LHX EXPRESSION IS ENTIRELY DEPENDENT ON THE 2.1 FUNCTION AND WE THINK THAT IT TAKE IT INDUCES THE EXPRESSION OF THESE GENES. AND HERE'S NK2 .1 EXPRESSION AND IN THIS CASE, WILD-TYPE MUTANT IS BELOW. THIS IS THE SONIC HGE HOG THIS, IS FUNCTIONED IN MANY DIFFERENT FORMS OF DIFFERENT ARENAS BUT 1 OF THEM IS IN PATTERN OF OF OTHER PROGENITOR ZONES. AND SONIC HEDGE HOG IN THE TELENCEPHALONS IS EXPRESSED IN THE PROGENITOR CELLS AND VENTRICULAR ZONES AS WELL AS IN SCATTERED NEURONS EARLY. BOTH OF THESE WILL BE IMPORTANT TO TODAY'S TALK. IF YOU DON'T HAVE NK 2.1 YOU DON'T EXPRESS CONIC HEDGE HOG, SO THIS MAKES THE TELENCEPHALON COMP AT THE PRESENT TIME FOR SONIC HEDGE HOG CONDUCTION OF EXPRESSION. NOW A MOUSE THAT LACKS NK2.1, HAS THE SLEEVER PROBLEMS IS MISSING LHX 6 AND 8 AND THE SONIC HEDGE HOG AND BASAL GANG RIA, IT HAS THE INFORMATION, BASICALLY TURNS INTO AN LGE. AND THAT CAN BE SEEN BY LOOKING AT MARKERS OF THE TRIAT UMKC AND GLOBEUS PALLETUS, THIS IS THE DOPAMINE 2 RECEPTOR. NICELY LABELED THE TRIAT UMKC AND NOT MUCH OF THE GLOBEUS PALLETUS, IN THE NK 2.1 YOU GET A HUGE STRIATUANDAT UM, LIKE WISE, THIS LABELS THE GLOBEUS PALLETUS AND THAT IS MISSING IN THE NK 2.1 MUTANT SO THIS DATA AS WELL AS MANY OTHER DATA BITS SHOW'S THERE A HOMEIOTIC CHANGE OF IDENTITY AND THE MGE, TURNS INTO THE LGE. NOW I'D LIKE TO TELL YOU WHAT THIS SONIC HEDGE HOG DOMAIN DOES IN THE MGE AND PREOPTIC AREA. WHAT ARE THE TYPES OF CELLS THAT ARE PRODUCED BY IT? FOR THIS, WE USE A MOUSE THAT WAS GENERATED IN ANDIE MCMAHON AND CLIFF TABBENS LAB, THIS IS A ON MOUSE IN WHICH THEY KNOCKED IN GFP, FUSED THE CREE INTO THE SONIC HEDGE HOG LOC OUST, IT--LOC OUST AND IT FUSIONS WITH THE CREE. AND THEN TO FOLLOW THE FATE OF THE CELLS THAT EXPRESS THIS CREE, WE CROSS WITH THE BETA GO LACTIC ACIDOSEISAICITIS REPORTER AND THEN WE ASSAY FOR BETA GALACTICICIDE ISOTOPE AND WE WILL REPORT CREE, AND GFP WHICH IS I MARKER OF THE CELLS THEMSELVES, BECAUSE IT'S A CREE GFP FUSION. SO THIS IS GFP EXPRESSION AND IT SHOWS YOU THE EXPRESSION OF SONIC HEDGE HOG, IT'S A BIT DARK IN YOUR PICTURE UNFORTUNATELY, I DON'T KNOW IF IT'S EASY FOR YOU, THE PROJECTOR--PROJECTION IS TO IMPROVE THIS, IN ANY CASES THESE ARE THE CELLS THEY ARE THE VENTRICULAR CELLS OF THE MGE AND THEN THE FATE OF THOSE CELLS IS TO PRODUCE THE NEURONS THAT ARE MIGRATE NOTHING THIS POSITION. CAN YOU SEE THAT OR IS IT 2 DARK? OKAY. SO WHAT ARE THE NEURONS MIGRATING OUT OF THIS CENTRAL MGE PROGENITOR ZONE. HERE'S A BRAIN ABOUT 2 DAYS OLDER THAN THE 1 I WAS JUST SHOWING. IT'S STAINED BY NK2 WENT 1 LABELING THE VENTICULAR ZONE CELLS AS WELL AS NUCLEUS IN THE UNDERLYING MANTEL OF THE MGE AND THAT'S THE GLOBEUS PAL ATUS. WHEN WE FIRST DID THIS WORK, WE THOUGHT THE GLOBEUS MALATUS MUST BE COMING FROM THE MGE BECAUSE IT LIFE ON LIES UNDERNEATH IT. BUT HAVING DONE THIS MAP, WE FOUND OUT WE WERE WRONG. SO THIS IS THE THE LABELING OF THE CELLS THAT HAD UNDERGONE COMBINATION AND THE VENTRICULAR ZONE OF THE MGE AND YOU CAN SEE HOW THEY'RE MIGRATING IN THIS DIRECTION AND WHEN YOU OVERLAY THIS WITH THE THE NK2.1, CAN YOU SEE THAT MOST OF THE NEURONS OF THE GLOBEUS PALATUS IN FACT, COME FROM THE MGE AND NOT THE THE DORSAL MGE, SO IT IDENTIFIED THE SOURCE OF THE GLOBEUS PALATUS. THEN WITH A MOUSE, ALSO MADE AN NK 2 WENT 1 CONDITIONAL ALLELE, WE ELIMINATED EXPRESSION ONLY IN THE VENTRAL MGE USING THE SONIC HEDGE HOG CREE, SO THIS IS A WILD-TYPE CONTROL USING NK2.1 RNA AS THE MARKER AND YOU CAN SEE THE EXPRESSION IN THE VENTRICULAR ZONE AND THE GLOBEUS PALATUS AND THEN IN THE MIDDLE WHERE WE DELETED IT AND YOU CAN SEE THAT THE GLOBEUS PALATUS STAINING IS ELIMINATED CONFIRMING THE ANALYSIS THAT THE VENTRAL MGE PRODUCES THE GLOBEUS PALATUS AND IT SHOWS THAT THE EXPRESSION OF THE MGE IS ESSENTIAL FOR THAT PROCESS. SO WITH REGUARD TO THE PROGENITOR DOMAINS IN THE BASIAN DECODER AT GANGLIA, WE IDEBTIFIED THE VENTAL MGE AND THE ZONE FOR MAKING MOST GLOBEUS PALATUS NEURONS. WE ALSO FOUND THAT THIS REGION, PRODUCES VERY FEW INTERNEURONS. ONLY ABOUT, I'D SAY 2% OF CORTICALE INTERNEURONS COME FROM THIS REGION. SO IT SEEM THIS IS ZONE IS PARTICULARLY IMPORTANT FOR PRODUCING NEURONS THAT REMAIN IN THE BASAL GANGLIA AND YOU'LL SEE FROM OTHER PARTS OF THE TALK WE THAT INTERNEURONS COME FROM THE DORSAL MGE. OKAY, SO TO SUMMARIZE THIS OLD DATA. NK2.1 SPECIFIES MGE IDENTITY, IT INDUCES LH6 AND 8 AND INDUCES SONIC HEDGE HOG. IT'S REQUIRED TO MAKE THE GLOBEUS PALATUS AND MAKE THE INTERDRIVE NEURONS, I DIDN'T SHOW YOU THE DATA BUT IF YOU MAKE THIS NOEL MUTANT YOU WOULD LOSE ABOUT 50% OF ALL CORTICALE INTERNEURONS. NK2.1 IS REQUIRED TO TURN ON SONIC HEDGE HOG HERE IN THE VENTRICULAR ZONE OF THE VENTRAL MGE AND PREOPTIC AREA. NOW LET ME TELL BUT WHAT LH-6 DOES AND THIS IS WORK DONE BY CHOW AND PIERRE CLAWNDINE AT E LAB. THE BOTTOM LINE IS AN LHX 6 MUTANT MUSE, YOU MAKE INTERNEURONS BUT YOU NEVER MAKE ALBUT HIV INFECTED PEOPLE POSITIVE OR SEMESTERATOSTATIN POSITIVE NEURONS THEY'RE DEPENDENT ON LHX 6 FUNCTION. WE USE AN ALLELE MADE BY REGEN RON WHICH THEY KNOCKED IN A PLACENTAL PHOSPHATASE OR P-LAP ALLELE INTO THE LHX 6 LOCUST DELETING MANY OF THE XONS MAKING THIS ANNUL ALLELE. BECAUSE THE P-LAP THEY WERE ABLE TO FOLLOW THE POSITION OF THE CELLS USING HISTOCHEMICAL STAINING FOR THE PHOSPHATASE. FIRST WE FOUND THAT THE GLOBEUS PAL ETICSUS WAS IN THIS MOUSE DESPITE IT WAS EXPRESSED ROBUSTLY IN MOST GLOBEUS PALLETUS NEURONS. AND WE ALSO FOUND THAT THEY ARE STILL PRODUCED AND STILL MIGRATE. SO THIS IS A HETEROZYGOTE AND A HOMOZYGOTE FOR THE P-LAP ALLELE. AND THIS IS THE CORTEX, THE PURPLE LINE OF CELLS IS A TAMPLEGGENTIAL MIGRATION OF THE INTERNEURONS GOING TO THE CORTEX AND YOU CAN STILL PRODUCE TONS OF THESE INTERNEURONS IN THIS MUTANT ALTHOUGH AS YOU NOTICE THEY ARE NOT MIGRATING AS FAST. AND THEN IF YOU--THESE ANIMALS WILL LIVE UNTIL ABOUT POST NEONATAL AND THEY DID THIS ANALYSIS AND THIS IS STAINING FOR PARVAL BUT HIV INFECTED PEOPLE AND THE WILD-TYPE AND THE MUTANT. IT'S A POSITIVE CELL AND THEN IF YOU STAIN FOR SOPHISTICATED MATOSTATIN IT'S THE SAME RESULT. SO THIS IS A TRANSCRIPTION FACTORS ARE VERY POWERFUL REGULATOR OF 2 OF THE MOST ABUNDANT AND INTERNEURON SUBTYPES OF OF THE NEOCORTEX AND HIPPOCAMPUS. PREVIOUS WITH OUR LHX 6 WORK, WE WORKED ON THE THE 8 MOUSE AND I WILL KEEP THIS VERY SHORT BUT3 BASICALLY, YOUNGUE FOUND THERE ARE CORTICALES FOUND IN THE STRIATUM AND IN OTHER REGIONS AND AS FAR AS WE CAN TELL RIGHT NOW, THE INTERCORTICALE NEURONS APPEAR NORMAL SO THIS IS JUST AN EXAMPLE OF THE POST NATAL TRIAT UMKC FOR CO LINE TRANSFER ACE, YOU CAN SEE THE NICE STAINING ON THE CELLS CELLS AND THIS IS THE MUSEUMITANT. THIS IS A 2 FOLD REDUCTION IN THE NUMBER OF COLONERGIC NEURONS AND THE OTHER SUBCORTICALE REGIONS. DESPITE THIS, 6 AND 8 HAVE SIMILAR STRUCTURES AT THE AMINO ACID LEVEL AND CO EXPRESSED IN ALMOST IDENTICAL PATTERN IN THE EMBRYONIC MGE AND THEN THEY HAVE VERY DIFFERENT FUNCTIONS LHX6 IS IMPORTANT AND THEY MIGRATE FOR SPECIFICATION, THE SUBTYPES WELL LHX 8 IS IMPORTANT FOR TRIATAL INTERNEURONS THAT ARE COLONERGIC. SO HERE'S A CORE CARD, I'LL BE USING INTERMITTENTLY DURING THE TALK AND LH6 MUTANT, THERE'S A STRONG PHENOTYPE, IN THE LHX MUTANT AND IN THE LHX8 MEWITANT THERE'S INTERNEURONS FOR THE CORTEX HERE. AND STRADDAL NEURONS DOES HAVE A DEFECT BUT NOT IN THE COLONERGIC BUT ONLY IN THE GABAergic STRIATUANDATTAL,OT OTHER POINT IT'S IMPORTANT FOR INTERNEURONS AND AT THE THE LEVEL OF THE GLOBEUS PALATUS, BOTH APPEAR TO PRODUCE A NORMAL APPEARING PALATUS, I DON'T KNOW ABOUT FUNCTION OF THE CELLS. SO THIS LEADS US TO THE QUESTION, WHAT HAPPENS IN THE DOUBLE MUTANT, SINCE THESE 2 TRANSCRIPTION FACTORS ARE CO EXPRESSED IN THE SAME CELLS, SAME PRONITOR CELLS. SO YOUNG U INITIATE THD PROJECT BY MAKING THE DOUBLE MUTANT AND WORK IN MY LAB WAS LED BY PIERRE FLAN DAN AND VOTE THEY HELPED PIERRE WITH THIS WORK AND THIS WORK WE JUST HEARD LAST WEEK IS FINALLY IN PRESS. THIS IT RECAPPITIULATES WHEN I'VE BEEN SAYING, THESE ARE CO EXPRESS INDEED THE BASAL GANGLIA AND THE BRACHIAL ARCHES THE EXPRESSION IS SOMEWHAT DIFFERENT BUT THEY'RE ALSO CO EXPRESSED IN THE MAXILLARY ARCH. AND NOW I'M GOING TO FOCUS ON SONIC HEDGE HOG EXPRESSION, NOT IN THE PROGENITOR CELLS BUT IN NEURONS. THIS IS AN AREA OF SONIC HEDGE HOG BIOLOGY THAT HASN'T BEEN EXPLORED VERY MUCH, IT'S PRIMARILY BEEN STUDIED IN THE SECTION OF THE CORD AS WELL AS IN THE FLOOR PLATE OF THE PROGENITOR ZONE. HERE I'M GOING TO TALK ABOUT SONIC HEDGE HOG EXPRESSION, I WILL ACTUALLY WALK OVER THERE. SO IN THE BASAL TELENCEPHALON THEREY'S A FLOOR LIKE STRUCTURE IN THE BOTTOM, WHERE YOU CAN SEE IT'S EXPRESS INDEED THE VENTRICULAR ZONE. AND YOU CAN SEE IT IN THIS SECTION, YOU GO FURTHER YOU DO HAVE IT BUT YOU DO HAVE THIS STRONG EXPRESSION, THIS STRONG EXPRESSION OF HEDGE HOG IS IN NEURONS, THE EARLY NEURONS OF THE THE MGE ARE FILL WIDE HAIR EXPRESSING CELLS AND YOU CAN TELL--CAN YOU SEE EASILY THERE'S A RATING OF THIS. SO HAVE YOU TON TONS OF SONIC HEDGE HOG NEURONS, AND ALMOST NO VENTRICULAR ZONE EXPRESSION AND CODLY IT'S THE OPPOSITE. THE UPPER PANELS ARE THE P-LAP REPORTER SHOWING WHERE LHX 6 IS EXPRESSED AND THIS IS SAYING WHERE LHX 8 IS EXPRESSED. SO, NOW, ON THE RIGHT, WE'RE LOOKING AT LHX 6 NULL, LHX8 NULL, DOUBLE MUTANTS AND IT'S EATS TO NOTICE THAT THE VENTRICULAR EXPRESSION OF SONIC HEDGE HOG IS UNEXPRESSED BUT THE NULL IS GONE. SO 3 A CLEAN PHENOTYPE THAT SHOWS THAT LHX 6 AND 8 TOGETHER ARE ESSENTIAL OF EXPRESSION IN SONIC HEDGE HOG IN NEURONS REMEMBER THAT IT'S DEPENDENT UPON LHX 2.1. SO IT'S VERY DIFFERENT IN TERMS OF WHAT TRANSCRIPTION FACTORS ARE SENSITIVE TO. TO SEE WHETHER THIS LOSS OF HEDGE HOG IN THE HEURONS HAS ANY EFFECTOT PROGENITOR ZONE, WE LOOKED AT SONIC HEDGE HOG RESPONSIVE GENES IN THE VENT LICKULAR ZONE OF THE MGE AND THESE ARE SOME EXAMPLES OF SOME GENES, PATCHED 1 AND NK6.2. AGAIN I'LL WALK OVER HERE BECAUSE IT'S SO COMPLICATED. SO THIS ISv/ CAUSE ILLEGALS SECTIONS WITHIN THE TEAL ENCEPHALON AND NK6.2, IS EXPRESSED ONLY IN THE DORSAL MGE AND THE THE PREOPTIC AREA. AND IF YOU LOOK AT PATCHED EXPRESSION, THIS EXPRESSION IS ALSO STRONGEST IN THE DORSE AT MGE. AND FOR MANY YEARS WORK E-PRESCRIBINGS IN OUR FIELD WERE MYSTIFIED BY THE FACT THAT THE STRONG HEDGE HOG RESPONSIVE GENES ARE FAR AWAY FROM HEDGE HOG IN THE THE VENTRICULAR ZONE OF PREOPTIC AREA. YOU'LL SEE THAT THIS HAS BEEN PARTLY ANSWERED BY THE FOLLOWING STUDY. NOW WE'RE GOING TO CONCENTRATEOT PATCHED EXPRESSION, WILD-TYPE AND MUTANT IF YOU COMPARE THE LEVEL OF PATCH EXPRESSION IN THIS DORSAL MGE, IN THE MUTANT WITH THE MUTANT, YOU CAN SEE THAT IT'S DOWN BY A FACTOR OF 2. SO THE LEVEL OF PAST EXPRESSION WHEN YOU REMOVE SONIC HEDGE HOG FROM THE NEURONS IN THE CELL HX6 AND DOUBLE MUTANT IS REDUCED. LIKE WISE, NKX 6.2 EXPRESSION IN THE DORSAL MGE IS PRODUCED BY A FACTOR OF 2, SUPPORTING THE MODEL THAT HEDGE HOG IN THE NEURONS IS CONTROLLING HEDGE HOG SIGNALING IN THEf#;ñ DORSAL MGE FAR FROM THE VENTRICULAR ZONE OF THE PREOPTIC AREA WHERE IT IS INITIALLY EXPRESSED. NOW LET'S TURN ORIGINAL ATTENTION TO THE GLOBE ALPALAT US AND THAT'S THE STRUCTURE HERE IN THE WILD-TYPE IN THE 6 AND 8 DOUBLE MUTANT THIS, IS THE WILD-TYPE AND THE LH6 AND 8 DOUBLE MUTANT, A GLOBEUS STRUCTURE FORMS BUT IT'S POORLY AGGREGATED CELLS SO THERE IS NOW A PHENOTYPE IN GLOBEUS PALATUS DEVELOPMENT WHEN YOU MAKE A DOUBLE MUTANT. CORTICALE INTERNEURONS, THIS IS THE CONTROL THESE ARE THE STREAM OF MIGRATING CELLS IN THE DOUBLE MUTANT AND THEN YOU STILL HAVE THE MIGRATING CELLS BUT IT'S A FACTOR OF 2 COMPARED TO THE LXX 6 MUTANT SO THERE'S A FURTHER WORSENING OF THE PHENOTYPE WHEN YOU REMOVE LHX AND 8. ONE OF THE REASONS WE THINK THAT THE--THERE'S A REDUCTION OF INTERNEURONS IS BECAUSE IN THE BASAL GANGLIA THERE'S A HUGE CLUSTER OF THESE CELL WHICH IS ACCUMULATE IN THE MGE, WHICH IS LABELED HERE AS ECTAUPIA, WE DON'T UNDERSTAND YET WHAT IS THE DEFICIT IN THE MATURATION AND MIGRATION THAT LEADS TO THIS ECTAUPIA BUT YOU CAN'T MISS IT. SO LHX 6 AND 8 TOGETHER FOR MIGRATION AND AND IN THE GENERATION OF THE GLOBEUS PALATUS. OT SCORE CARD, THIS IS THE PALM WITH THE DOUBLE MUTANT, THE INTERNEURON PHENOTYPE IS WORSE THAN THE LHX MUTANT AND SHOWING THAT IT IS IMPORTANT IN INTERCORTICALE DEVELOPMENT AND THERE'S NO PHENOTYPING IN THAT NULL MUTANT AND IN THE TRIATAL INTERNEURONS, THIS IS THE COMBINED OF DEFECTS IN GABBA FEIGNAL CO LINE INTERNEURON AND NOW WITH THE DOUBLE MUTANT WE HAVE THE GLOBEUS PALATUS PHENOTYPE AND MOST IMPORTANTLY FOR THIS TALK, SONIC HEDGE HOG EXPRESSION IN NEURONS OF THE MGE IS LOST. NOW WE CAN'T PARSE THE ROLE OF HEDGE HOG EXPRESSION THAT'S LOST IN THIS EXPRESSION, AND FUNCTIONS 6 AND 8, DO HAVE CONTRIBUTED TO THIS OVERALL PHENOTYPE WITHOUT DOING ANOTHER EXPERIMENT SO YOU'LL HAVE TO SIT THROUGH ANOTHER EXPERIMENT. D. SO HERE WE EAR, NK2.1 TURNS ON SONIC HEDGE HOG ON THE VENTRICULAR ZONE AND TURNS ON LHX6 AND 8 WHICH THEN TURN ON SONIC HEDGE HOG IN THE NEURONS. SO NOW I'M GOING TO DESCRIBE AN EXPERIMENT WHERE WE TESTED SONIC HEDGE HOG IN THE NEURONS AND WHAT IT DOES BY DOING A MUTE O GONE SIS OF HEDGE HOGS IN THESE CELLS. SO WHAT IS THE FUNCTION OF SONIC HEDGE HOG IN THESE EARLY BORN NEURONS OF THE MGE, THE CELLS THAT ARE MISSING ALL THIS NICE HEDGE HOG THAT'S PRESENT IN THE WILD-TYPE. ANDIE MCMAHON MADE ANOTHER IMPORTANT ALLELE THAT HE MADE AVAILABLE TO US AND THAT IS A CONDITIONAL ALLELE OF SONIC HEDGE HOG IN WHICH XON 2 WAS LOCKED BY CITES AND IT WAS MEDIATED BY CREE EXPRESSION EXON TO MAKE A NULL ALLELE AND THEN WE HAD TO FIND A CREE THAT WOULD BE APPROPRIATE TO REMOVE HEDGE HOG FROM THE NEURONS BUT LEAVE IT ALONE IN THE VENTRICULAR ZONE OF THE MGE AND POA. AND LUCKILY WITH OUR NIMH GRANT, WE MADE A DEAL, 1, 2, CREE, AS I MENTIONED THIS IS EXPRESSED IN THE LGE AND MGE AND WE DORPHED IN THE LAB WITH ENHANCERS THAT ARE DRIVING NORMAL EXPRESSION, THIS ENHANCER IS AN INTERGENE ENHANCER BETWEEN THE 1 AND 2 GENES AND GREG POTTENER MY LAB MADE THIS MOUSE. AND WE FOUND IT NICELY LABELS THE NEURONS OF THE MGAND AND LGERONTOLOGYSTS BUT DOESN'T TOUCH THE VENTRICULAR ZONE EITHER. MAKING IT APPROPRIATE FOR THIS EXPERIMENT. WE WANT TO REMOVE SONIC HEDGE HOG BUT LEAVE IT ALONE IN THE VENTRICULAR ZONE. SO HERE, PIERRE DID THAT EXPERIMENT. HERE IS THE SONIC HEDGE HOG EXPRESSION IN THE NEURON AND THE VENTRICULAR ZONE OF A WILD-TYPE ANIMAL AND THIS IS THE EXPERIMENTAL ANIMAL WHERE WE REMOVE SONIC HEDGE HOG ONLY FROM THE NEURONS AND LEFT THE EXPRESSION ALONE IN THE VENTRICULAR ZONE SO OUR CONTROL EXPERIMENT SHOWED US THAT WE'VE GOT LUCK SCHESUCCEEDED. IN MAKE THANKSGIVING ANIMAL, SO WHAT DOES IT DO IN NEURONS. THE FIRST THING THAT PIERRE TESTED WAS, IS THERE A CHANGE OF HEDGE HOG SIGNALING IN THE OVERLYING PROGENITOR CELLS AND THE ANSWER IS YES. IT'S A BIT SOWBTLE SO I'LL COME OVER HERE AND WORK THROUGH THIS. SO HERE WE USE PATCHED WHICH IS A HEDGE HOG RESPONSIVE GENE AND LET'S LOOK AT PATCHED HERE IN THE DORSAL MGE OF THESE WILD-TYPES, AGAIN IT'S HIGHEST IN THE DORSAL MGE AND DOWN HERE IN THE VENTRAL MG AND THE POA AND YOU CAN SEE IN THIS MUTANT THE VENTRAL MGE AND THE PATCH LOOKS PRETTY MUCH THE SAME WHERE THE DORSAL IS REDUCED BY A FACTOR OF 2 AND SHOWING THAT HEDGE HOG EXPRESSION IN THESE NEURONS IS SIGNALING TO THE OVERLYING PROGENITOR CELLS. THIS IS HOW WE INTERPRET IT, SO NEURONS ARE REGULATING PROGENITOR CELLS. ANOTHER NICE HEDGE HOG MARKER IS THE TRANSCRIPTION FACTOR GLEE 1 AND NK2 IS EXPRESSED IN THE DORSAL NG E AND IN THE MUTANT AREA THE PREOPTIC IS FINE BUT THE DORSAL HAS ABOUT A FACTOR OF 2 DECREASED EXPRESSION. SO NOW THE MODEL HAS EVOLVE TO THIS POINT. NX2.1 REGULATES HEDGE HOG IN THE VENTRICULAR ZONE OF THE VENTRAL REGIONS AND IT TURNS ON LHX 6 AND 8 TO TURN ON HEDGE HOG IN THE NEURONS WHICH THEN REGULATES HEDGE HOG SIGNALS IN THE DORSAL REGIONS. AND WE'RE THINKING THIS IS THE WAY THAT YOU CAN HAVE A STRUCTURE THAT'S GROWING LARGER AND LARGER OVERTIME AND EVOLUTION THAT NEEDS HEDGE HOG TO CONTROL THE PROPERTIES OF THE PROGENITOR ZONES BUT IF YOU DON'T HAVE HEDGE HOG EVERYWHERE IN THE REGION YOU NEE TO HAVE A PRODUCED IN ANOTHER SIGHT SO THIS ALLOWS THE HEDGE HOG AND REGIONS THAT ARE FAR FROM THE HEDGE HOG IN THE VENTRICULAR ZONE. AND AS SUCH, THE PARTS OF THE DEVELOPING NG E IN THIS MUTANT ARE THE ROST ILLEGALS REGIONS SO THIS IS A SECTION THROUGH THE ROST ILLEGALS PARTS OF IT THROUGH THE ENCEPHALON THIS IS WILL BE WITH THE ENSEPTIN IS, AND IT'S FAR WHERE THE PREOPTIC AREA IS, I DON'T KNOW HOW MANY MILLIMETERS BUT THIS IS--THIS IS QUITE FAR AWAY AND IN THE WILD THIS, IS ALL WILD-TYPE ON THE LEFT COLUMN AND YOU CAN SEE THE NX2.1 EXPRESS INDEED THE TINY LITTLE DOMAIN IN THIS REGION SHOW THANKSGIVING'S A REG FORWARD OF THE MGE IN THIS REGION BUT IF YOU MOVE SONIC HEDGE HOG FROM THE NEURONS CAN YOU SEE THAT THE 2.1 DOMAIN IN THE PROGENITOR ZONE IS NOT DETECTABLE SO THE ROSTRIEL AREA IS IN THE SEPTUM IS DEPENDENT ON HEDGE HOG SIGNALING COMING FROM THESE NEURONS. FURTHER MORE, CORTICALE DEVELOPMENT IS DISRUPTED. 21 ANIMALS SEEMATOSTATIN AND TOI AND FOUND ABOUT A 40 FOLD, 40% REDUCTION IN THE NUMBER OF PARALBUT HIV INFECTED PEOPLE SEMESTERATOSTATIN AND POSITIVE INTERNEURONS IN IF MUTANT AND NPY INTERNEURONS WERE UNCHANGED, AND PIERRE FOUND THAT INTERNEURONS WERE REDUCED MORE THAN EARLY BORN INTERNEURONS. THIS IDEA THAT HEDGE HOG EXPRESSION IN THEEURONS AND ROSTRIEL REGIONS MAY NOT ONLY HAVE A SPACIAL CONTROL OF THE ROST RIEL DORSAL MG BUT A TEMPORAL ROLE IN PROMOTING AND MAINTENANCE OF THE STEM CELLS SO THEY CAN PRODUCE LATE INTERBORN NEURONS. THE SISTER HERE SHOWS THE EFFECT ON THE EARLY BORN INTERNEURONS WITH THE REDUCTION OF THE PARALBUT HIV INFECTED PEOPLE AND NODE DETECTABLE EFFECT ON THE MPY POSITIVE INTERNEURONS. NOW TO FINISH I'LL TALK ABOUT A LITTLE BIOCHEMISTRY. HOW DO LHX 6 AND 8 REGULATE SONIC HEDGE HOG EXPRESSION. FORTUNATELY FOR US, DOUG EPISTEIN'S LAB HAS BEEN WORKING CAREFULLY ON SONIC HEDGE HOG ENHANCERS AND IN 1 OF HIS PAPERS HE REPORTED AN ENHANCER THAT'S EXPRESSED IN THE NEURONS OF THE EMBRYONIC MGE, AND THIS IS THE SEQUENCE OF THAT ENHANCER SO WE PUT THIS ENHANCER THROUGH A BIT OF INFORMATICS AND FOUND A CANDIDATE LHX BINDING SITE WHICH IS UNDERLINES IN RED HERE AND WE GENERATED ELECTROFORETICSIC MOBILITY ASSAY AND PROBE THAT'S THE SIZE OF THIS BOX REGION AND TESTED WEATHER OR NOT LHX 6 AND 8 COMBINED TO THIS ENHANCER INVITRO. SO WE PLANNED THESE EXPERIMENTS, HE EXPRESSED RECOMBINANT LH6 AND 8 PRACTICES PAIR FRIDAY THE NUCLEOTIDES LE I AND THEN DID DNA BINDING ASSAYS WITH THE MOBILITY SHIFT ANALYSIS. AND SO I'LL WE JUST DECIDED TO KEEP IT SIMPLE. THIS LANE IS THE EXTRACT ITSELF, BOUND TO THE PROBE. AND THERE ARE PROTEINS THAT BIND TO THE PROBE AT THESE 2 POSITIONS, OR IF WE HAVE AN EXTRACT THE RECOMBINANT LHX 8 IN IT, WE GET THIS NICE BIG BAND THAT'S SEPARATE FROM THESE 2 ENDOGENOUS PROTEINS. IF WE THEN ADD UNDER A HUNDRED FOLD EXCESS OF PROBE, WILD-TYPE PROBE TO IT, WE CAN ELIMINATE THE BINDING OF ALL 3 PROTEINS GO AWAY. THIS IS WHERE WE HAVE AN ANTIBODY TO THE FLAG OR TAGGED TO THIS RECOMBINANT PROTEIN AND WE CAN GET A NICE SHIFT, A SUPER SHIFT OF THIS COMPLEX, AND THEN THERE'S AN EXPERIMENT WHERE WE TOOK THE PROBE AND MUTATED THE LHX 6 CORE BINDING SITE TO I THINK ALL CYTOSIGNS. NOW OUR LHX PROTEIN NO LONGER BINDS IT BUT THESE 2 ENDOGENOUS PROTEINS THEY DON'T6í CARE, THEY STILL BIND SO WE DID A LOT OF CONTROLS AND WE HAD GOOD EVIDENCE NOW THAT AT LEAST INVITRO, LHX 6 LHX 6, TO THIS CORE BINDING SITE OF THE SONIC HEDGE HOG ENHANCER THAT IS EXPRESS INDEED THE NEURONS OF THE MGE AND AS A FINAL ASSAY, WE TEST INDEED MGE CELLS THE REAL THING WHETHER OR NOT THE REDOM BIN ANT LHX 6 AND 8 CAN REGULATE THIS ENHANCER SO WE TOOK EXPRESSION FACTORS WELL WELL AS A SONIC HEDGE HOG CANCER DRIVING THE CHERRY REPORTER AND CO TRANSFECTED THESE INTO THE MGE CELLS, MEASURING THE ABILITY OF LH6 AND 8 TO INCREASE THE AMOUNT OF M-CHERRY PROTEIN. SO THESE NICELY SHOW THAT LHX 6 AND 8, ARE ABLE TO INDUCE THE AMOUNT OF M-CHERRY PRODUCED OFF OF THE SONIC HEDGE HOG ENHANCER THIS GUY HERE IS WITHOUT ANY ADDED LH6 OR 8 AND THEN, THIS IS WITH THE WILD-TYPE ENHANCER AND WE MADE THE ENHANCER WITH THE MUTANT LHX BINDING SITE AND ASK WHETHER OR NOT THESE TRANSCRIPTION[4z FACTORS WHETHER THESE TRANSCRIPTION FACTORS CAN NOW ACTIVATE EXPRESSION AND THIS IS WITH THE MUTATED BINDING SITE AND AS YOU CAN SEE WE DON'T GET ANY DETECTABLE ACTIVATING OF THAT BACKGROUND. SO BOTH INVITRO, DNA BINDING AS WELL AS IN VIVO IN THE CORRECT CELLS, TRANSCRIPTION FACTOR ASSAYS, WE HAVE GOOD EVIDENCE THAT LHX 6 AND 8 ARE ABLE TO ACTIVATE THIS ENHANCER. WE DON'T HAVE GOOD ANTIBODIES FOR LH6 AND 8 SO WE DON'T HAVE FORMED PRECIPITATION TO SEE WHETHER OR NOT THEY ARE PRESENT ON THE SONIC HEDGE HOG ENHANCER IN VIVO. IN THE PRIMARY PROGENITORS THE STEM CELLS IN THE VENTRICULAR ZONE EXPRESS MKX AND DLX FUNCTION, IT DOES AN ENTIRELY DIFFERENT FUNCTION OF PROCESSES AND REGULATES LEVEL OF GAD, REGULATES MIGRATION THESE CELLS LIVE IN THE SUBVENTRICULAR ZONE, IF YOU BECOME A ISHT NEURON YOU LEAD THE EXPRESSION ON YOU TURN ON LH6 AND 8 AND THEN YOU HAVE TO TURN OFF NK2 WENT 1. WE'RE JUST BEGINNING TO UNDERSTAND HOW THAT HAPPENS BUT I'M NOT IN THE POSITION TO TELL YOU HOW THAT OCCURS. IF YOU DON'T TURN OFF NK2 WENT 1, YOU DO NOT BECOME A TRIATAL INTERNEURON OR THE GLOBEUS PALATUS. THEN WITHIN THE SUBVENTRICULAR ZONE, EXPRESS DLH AND 6 AND 8. AND IF YOU BECOME A CORTICALE INTERNEURON YOU NEED TO TURN ON ALL THESE TRANSCRIPTION FACTORS AND RECEPTORS AS WELL AS SONIC HEDGE HOG. AND IF YOU'RE AN MGE TYPE OF CORTICALE INTERNEURON PROGENITOR, YOU MAKE THE ALBUT HIV INFECTED PEOPLE CELLS AND THE SEMESTERATOSTATIN CELLS AGAIN TOTALLY DEPENDENT ON LHX 6 FUNCTION. WE RECENTLY PUBLISHED A PAPER SHOWING THAT THESE 2 CYTOKINE RECEPTORS CXC4 AND RD1, ARE PART OF THE PROGRAM THAT ARE ESSENTIAL FOR DRIVING THESE PARTICLES TO THE CORRECT LAMINA AND THESE RECEPTORS ARE DEPENDENT UPON LHX 6 FUNCTION AND DEPEBBLEDDING ON WHAT LHX 6 DOES. FINALLY IF YOU INCORPORATE THE PALLET, YOU HAVE TO GET NK2.1 TO STAY ON AND THEN HAVE YOU A GLOBEUS PALATUS NEURON SO THE SWITCH OF TURNING OFF NK 2.1 IS CRITICAL IN THE CELL FATE DECISIONS. , THEREFORE EARLY SONIC EXPRESSION WHICH IS DEPENDENT UPON NK2 WENT 1 IN THE VENTRICULAR ZONE, IS IMPORTANT FOR THE MGE AND POA, ON THE OTHER HAND, LATE SONIC HEDGE HOG EXPRESSION IN IT 6 AND 8 IS IMPORTANT FOR PATTERNING THE DORSAL MGE AND THE DORSAL MGE IS THE PART THAT EXTENDS ROSTERALLY IN THE SEPT UM AND IT HAS OBVIOUS IMPORTANCE FOR NEUROPSYCHIATRIC ILLNESS. I'D LIKE TO CONCLUDE NOW. I THINK I MENTIONED MOST OF MY COLLABORATORS. I'LL LEAVE THIS SLIDE ON SHOWING HOW GOOD THE BASEBALL TEAMS AND ARE BASEBALL TEAM IS IN SAN FRANCISCO AND I'LL PUT THE HARD HAT ON FOR THOSE QUESTIONS. THANK YOU. [ APPLAUSE ] >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> THE QUESTION IS: HOW CAN FIXENATE CONTROL SUCH OVERLAPPING YET DIFFERENT PATHWAYS? WE DON'T KNOW. WE DON'T KNOW. THERE ARE REALLY--THE THE RNAs ARE IN THE SAME CELLS AS FAR AS WE CAN TELL. WE DON'T HAVE GOOD ANTIBODIES SO WE DON'T KNOW THAT LEVEL OF ANALYSIS WHETHER THE PROTEINS ARE IN DIFFERENT CELL TYPES. AT THE LEVEL OF THE SONIC HEDGE HOG ENHANCER WE HAVE NO EVIDENCE FOR A DIFFERENCE THAT'S REALLY CLEAR IN THE ACTIVITY OF THE 2 PROTEINS. YEAH, WE DID SINGLES AND WE DIDN'T FIND ANY SYNERGY, WE JUST FOUND EVIDENCE FOR REDUNDANCY. WE DID FIND SOME ACTIVITY WITH LHX ON THE ENHANCER BUT I DON'T TRUST THE ASSAYS AT THAT LEVEL OF RESOLUTION. TOM? >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> I THINK WE'RE READY TO TRY. I THINK YOSHIKI'S WORK IS PROBABLY THE BEST WHERE HE CAREFULLY LOOKS AT A LOT OF MY PAPERS AND A LOT OF OTHER PEOPLE'S PAPERS TO IDENTIFY THE GENETIC RECIPES REQUIRED IN VIVO AND HE THEN, USING SOLUBLE FACTORS BUT TITRATING THEM AND USING REDIALS OF THE TRANSCRIPTION FACTORS I'VE BEEN TALKING ABOUT HAS DEVISED QUITE GOOD TECHNIQUES, LARGE IN MOUSE CELLS BUT AUSIN SOME HUMAN CELLS TO MAKE CORTEX AND TRIAT UMKC AND MGE TYPE OF CELLS. WE HAVE BEEN COPYING HIM AND HE'S ACTUALLY A COLLABORATOR AND HELPS US WITH OUR WORK AND I THINK WE'RE PRETTY GOOD SHAPE FOR NOW. PRETTY GOOD SHAPE WITH HUMAN ESLs TO MAKE MGE TYPE INTERNEURONS AND AS YOU MAY KNOW, WORK WITH ARE--ADMINISTRATIVEATURE O'S AND SCOTT, AND WE HAVE A GRANT WHERE WE ARE USING MOUSE MGE CELLS TO TREAT MOUSE DISEASES. AND WE FOUND PRETTY GOOD EVIDENCE THAT TRANSPLANTATION OF MOUSE MGE CELLS INTO THE POST NATAL CORTEX OF AN EPILEPTIC MOUSE GREATLY REDUCES THEIR SEIZURES AND LIKE WISE ARNOLD HAS EVIDENCE THAT THESE CELLS TRANSLATE INT A PARKINSONIAN STRIATUANDAT UM CAN IMPROVE THAT. SO WE'RE INTERESTED IN THE POSSIBILITY OF CELL$iBASEDDED THERAPIES FOR A NUMBER OF NEUROLOGICAL AND PSYCHIATRIC ILLNESSES. MARK? OLD FRIEND. >> [INAUDIBLE RESPONSE FROM AUDIENCE ] >> I DON'T REMEMBER HOW MANY KB FROM THE PROMOTER. ACTUALLY I DON'T REMEMBER WHICH 1. I DON'T REMEMBER. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> I BELIEVE THAT IN MOST OF OUR ANALYSIS, WE USED AS OUR CONTROL THE HETEROZYGOTE ALLELE SO THAT WE CAN AT LEAST ACCOUNT FOR WHERE THAT IS A CONTRIBUON. YEAH, WE WEREN'T WORRIED ABOUT IT. >> SO HAVE YOU TRIED TO RESCUE THE NK PHENOTYPE BY EXPRESSING THE DOWN STREAM TARGET SAY [ PH KD [INDISCERNIBLE]R-- >> WE'RE EXPLORING, SO WE'RE GOING TO TAKE ADVANTAGE OF THE PHENOTYPE AND OUR MGE TRANSPLANTATION ASSAY, SO NOW WHAT WE CAN DO IS WE CAN TRANSPLANT THE MGE FROM THE THE--LET'S SAY LHX 6 MUTANT INTO A WILD-TYPE CORTEX AND DANIEL FOUND THAT INDEED THOSE CELLS CANNOT MAKE THE SOMATOSTATIN BUT THEY CAN MAKE THESE CELLS, THEY WILL USE THAT ASSAY TO TRY TO RECONSITUTE THE CELLS BY PUTTING IN DIFFERENT COMPONENTS. SO WE'RE GOING TO EXPLORE SOME OF THE OTHER TRANSCRIPTION FACTORS LIKE ARX AND MATH B AND THOSE ARE DOWN REGULATE INDEED THOSE CELLS TO SEE WHAT THESE COMPONENTS DO FOR THAT PHENOTYPE. >> BUT YOU CAN ALSO TYPE BY PUTTING SOME [INDISCERNIBLE] TOGETHER AS WELL. >> SURE, THAT'S ANOTHER GOOD WAY TO DO IT. YEAH? NO AJ WAS NEXT. YEAH? >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> YEAH, THAT'S A GOOD QUESTION. FIRST OF ALL WHY IS IT ONLY HALF REDUCTION OF THE HEDGE HOG RGETS. IT MAY BE IN THE CONDITION OF THE MUTANT WHERE WE MAYBE DIDN'T KNOCK IT OUT EARLY ENOUGH IN THE CELLS BUT IN THE LHX 6 AND 8 MUTANT IT'S THE SAME. SO IT MAY BE THAT OTHER MECHANISMS INDUCE THAT AND I DON'T KNOW WHAT IT IS AT THIS POINT AND THEN THE TRAFFICKING OF THE HEDGE HOG PROTEIN FROM THE NEURONS TO THE VENTRICULAR ZONE IS SOMETHING WE WOULD LOVE TO EXPLORE. OUR SIMPLEST IDEA IS THAT THE VENTRICULAR ZONE HAS THE RADIAL GLEEL PROCESSES THAT GOES RIGHT THROUGH THE NEURONS AND THOSE ARE THOUGHT--IT LIKELY THEY CAN JUST BIND TO THOSE PROCESSES AND THEREBY SIGNAL BACK TO THE NUCLEUS. YEAH. >> I BELIEVE SO, I DON'T THINK WE SAW ANYTHING INTERESTING AT THE LEVEL OF CORTEX VERSES HIPPOCAMPUS, I THI THE--IT'S AMAZING BUT THE SAME RULES SEEM TO HOLD FOR CORTICALE INTERNEURONS AND HIPPO HIPPOCAMPAL NEURONS LARGELY NOT ENTEARLY. --I DON'T REMEMBER.4pL YEAH, BY THAT 40%. YEAH? >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> SAY AGAIN? >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> NO, IT POINTS TO THE FACT TAKEN--THEYg3 THERE'S A TRANSCRIPTION FACTOR THAT'S UPSTREAM OF NK2 .1 THAT'S DRIVING EXPRESSION OF FEEDBACK NK2.1 PROTEIN IS NOT NEEDED FOR MAINTENANCE. SO, THERE'S A LOT OF WAYS TO SQUIRREL AROUND GETTING TO ANSWER THAT QUESTION, BUT WE DON'T KNOW WHAT THAT TRANSCRIPTION FACTOR IS, IT'S EVIDENCE THAT FIXED A GENE, CALLED FIX 3, 2 K1 IS IMPORTANT IN CENTRAL, FOREBRAIN DEVELOPMENT AND CAN INDUCE SONIC HEDGE HOG IN THAT REGION AND LIKE WISE FGF EXPRESSION IS ESSENTIAL FOR INDUCTION OF THE MGE AND THE TRANSCRIPTION FACTORS OF FGF SIGNALING ARE NOT FULLY WORKED OUT YET, SO, THERE'S MORE COMPLEXITY THERE THAT'S ENOUGTO EXPLAIN THAT RESULT. YEAH? >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> YES THAT'S RIGHT AND I'LL MENTION IT NOW SO WITH YONUG, GLUE, WE EXPRESSION THE FACTORS, THE LDB-1 AND IN OUR BIOCHEMICAL EXPERIMENTS, ALL THE EMSAN FRANCISCO BINDING WAS DONE IN THE PRESENCE OF LDB 1 AND IN THE ABSENCE WE COULDN'T FIND GOOD BINDING. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> YES. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> THE QUESTION WAS: WHY DO WE--HOW CAN WE TOTALLY EXPLAIN THE--NO, ANOTHER WAY. WELL'S A REDUCTION IN THE SONIC HEDGE HOG RESPONSE IN THE GEANAL EXPRESSION OF THE DORSAL MGE, IS THAT DUE TO GENE EXPRESSION OR COULD TED CELLS BE DYING? AND WE ACTUALLY LOOKED AT CELL DEATH AND AT EARLY TIME POINTS WE DON'T SEE INCREASED NUMBERS FOR APOPTOTIC CELLS BUT INTERESTINGLY THE TIME THAT'S IRRELEVANT FOR YOUR QUESTION, AT THE TIME WHEN THE INTERNEURONS ARE MIGRATING THERE IS A LOT MORE CELL DEATH AND 1 OF THE POSSIBLE REASONS THAT--1 OF THE POSSIBLE FUNCTIONS OF HEDGE HOG, LATE IN HEDGE HOG FUNCTION IS NOT JUST IN PROLIFERATION OR PATTERNING BUT COU BE IN SURVIVAL OF THOSE SECONDARY PROGENITOR NEURONS. WE'RE TRYING TO CROSS THOSE MICE NOW WITH 1 OF THESE CELL DEATH CAUSING GENES IN MUTANTS AND THAT'S TO RESCUE THEM WHEN IT'S BACK, THAT RESCUES THE INTERNEURON TYPE. >> YES. THERE'S ANOTHER BIG ZONE OF IRPT NEURON PRODUCTION CALLED THE DORSAL CGE AND THAT'S WHERE A LOT OF THE MPY CELLS ARE PRODUCED. NOT ALL. THE EARLY BORN NPY CELLS ARE PRODUCED FROM THE NG BUT THERE'S WHOLE OTHER SET PRODUCED FROM THE CGE AND SO, WE WOULD EXPECT WITH OTHER CGE MARKERS WE WOULD SEE PRESERVATION OF THAT. WE HAVEN'T DONE THAT YET. ABOUT 40% OF CORTICALE IRPT NEURONS COME FROM THE CGE AND 60% ROUGHLY FROM THE MGE, OR MAYBE 50 FROM THE MGE AND ANOTHER 10% FROM THE POA. >> THANKS JOHN. >> [ APPLAUSE ]