>> WE'LL GET STARTED. HE BECAME ASSOCIATE DIRECTOR ALSO AT THE NORTH CAROLINA CENTER FOR MENTAL HEALTH RESEARCH, AND WAS A PROFESSOR AT THE DEPARTMENT OF CELL AND MOLECULAR PHYSIOLOGY. IN 2010 HE WAS ACTUALLY DRAWN AWAY AND APPOINTED TO HIS CURRENT POSITION AT GEORGE WASHINGTON UNIVERSITY WHERE HE IS TODAY. SO DR. LAMANTIA'S WORK HAS REALLY FOCUSED ON LOOKING AT A LOT OF ASPECTS OF NEURODEVELOPMENT. AS LABORATORY CURRENTLY EXPLORES GENETIC MOLECULAR MECHANISMS OF EARLY FOREBRAIN DEVELOPMENT, STUDYING SIGNAL AND TRANSCRIPTIONAL REGULATION FOR STEM CELL IDENTITY AND THE ROLE OF FOREBRAIN DEVELOPMENT REGULATORY GENES. BEHAVIORAL AND PSYCHIATRIC DISEASES. THIS INCLUDES THINGS LIKE SCHIZOPHRENIA AND AUTISM. IN ADDITION, HIS LABORATORY STUDIES MOLECULAR MECHANISMS THAT SPECIFY NEUROSTEM CELLS IN THE EMBRYONIC AS WELL AS ADULT NERVOUS SYSTEM AND HE'S FOCUSING ON THE SPECIFIC CASE OF NEUROSTEM CELLS -- A PLACE THAT'S NEAR AND DEAR TO MY OWN HEART. GIVEN THE ASSOCIATION BETWEEN OLFACTORY DYSFUNCTION AND NEURODEGENERATION SUCH AS ALZHEIMER'S AND PARKINSON'S, THIS APPROACH ACTUALLY PROVIDES VERY INTERESTING WAY POTENTIAL MODEL FOR STUDYING DEGENERATION AND POTENTIAL REPAIR ASSOCIATED WITH NEURAL LOSS OF DYSFUNCTION IN THESE DISORDERS. DR. LAMANTIA'S SCIENTIFIC SERVICES ARE ALSO MANY. HE'S APPOINTED TO VARIOUS EDITORIAL BOARDS. HE'S BEEN CHAIR AND MEMBER OF NUMEROUS NIH STUDY SECTIONS AND CURRENTLY INCLUDES THE EXTRAMURAL ADVISOR TO THE INTRAMURAL RESEARCH PROGRAM AT NNDS AND HIS PRESENTATION TODAY WILL DISCUSS THE SPECIFICATIONS OF OLFACTORY AND FOREBRAIN STEM CELLS DURING EARLY EMBRYONIC DEVELOPMENT. IT'S A PLEASURE TO HAVE YOU HERE AND WE LOOK FORWARD TO IT. >> THANK YOU FOR THAT REALLY LOVELY INTRODUCTION. IT'S REALLY NICE TO BE INTRODUCED BECAUSE YOU RECOGNIZE THAT PEOPLE THINK MUCH MORE OF YOU THAN YOU THINK OF YOURSELF. SO IT'S ALSO VERY NICE TO BE HERE AND IT'S VERY NICE TO BE ABLE TO TAKE JUST A SHORT TRAIN RIDE AND COME TO THIS REALLY REMARKABLE ENVIRONMENT FOR NEUROSCIENCE RESEARCH. SO I THANK YOU ALL FOR THE OPPORTUNITY. WHAT I'M GOING TO TELL YOU ABOUT TODAY IS IN A SENSE WHAT MAY BE A SURPRISING CONNECTION TO SOME OF YOU BUT IS NEVERTHELESS A REALLY IMPORTANT CONNECTION AND HATS THE CONNECTION BETWEEN NOSES AND FOREBRAIN AND NEUROSTEM CELLS. I THINK WHAT I'M GOING TO TRY TO CONVINCE YOU IS THAT THE MECHANISM BY WHICH THE STEM CELLS THAT MAKE THE SENSORY EPITHELIUM IN THE NOSE AND THE STEM CELLS THAT ARE GOING TO CONSTITUTE BOTH THE COMPONENTS OF THE BASAL FOREBRAIN AS WELL AS THE NEOCORTEX. BUT THOSE STEM CELLS ARE ACTUALLY SPECIFIED IN REMARKABLY PARALLEL MANNERS WITH SOME OF THE SAME MECHANISMS AND SOME OF THE SAME MOLECULES INVOLVED. SO WHEN WE THINK ABOUT THIS EMBRYONICALLY IT'S NOT HARD TO APPRECIATE THAT IS THE CASE BECAUSE IF WE LOOK AT AN E9 EMBRYO, WE SEE THIS BLUE PATCH HERE I COLORED IN BLUE, THEY DON'T COME BLUE, IS ALSO THE REGION OF THE EMBRYO THAT'S GOING TO BECOME THE OLFACTORY. AS WE GO THROUGH DEVELOPMENT WE SEE THIS DIFFERENTIATES AND BEGINS TO FRO. THE EPITHELIUM IS DEEP IN THE NASAL STRUCTURES HERE. BUT AT THE SAME TIME, AND YOU CAN SEE THAT THIS STARTS WITH A SIMPLE EPITHELIUM OPPOSED TO THESE PARENCHYMAL CELLS, CONTINUES TO DIFFERENTIATE SO YOU HAVE ADVANTAGE NATION AND EPITHELIUM OF EPITHELIUM BEGINNING TO FOLD OUT AND POCKET IN. FINALLY YOU HAVE THIS VERY COMPLEX STRUCTURE THAT HAS WITHIN IT THE PRIMARY SENSORY NEURONS THAT DETECT ORDOR -- AT THE SAME TIME THIS IS HAPPENING, UP HERE RIGHT ACROSS THE ACROSS THE WAY FROM THE NASAL REGION IS THE FOREBRAIN. AND THE FOREBRAIN IS GOING TO CONTINUE TO DIFFERENTIATE RIGHT NEXT TO THE FIELD WHERE THE NOSE IS DIFFERENTIATING. THIS IS NOT JUST A CASUAL RELATIONSHIP BECAUSE WHAT WE SEE IS THAT WHAT BASICALLY YOU HAVE AT THESE EMBRYONIC STAGES IS YOU HAVE TWO EPITHELIA, THE SURFACE ECTO DETERMINE WHICH IS GOING TO IMPART EPITHELIUM, IT'S GOING TO NEURALLIZE, THERE ARE GOING TO BE STEM CELLS THAT GENERATE ULTIMATELY THE NEURON TYPES IN THE PERIPHERY. AT THE SAME TIME LET'S AN EPITHELIUM HERE, IT'S OPPOSED TO THE SAME MESENCHYME AND THIS EPITHELIUM IS GOING TO ACTUALLY BECOME ALL OF THE FOREBRAIN. SO BASICALLY YOU HAVE A VERY LIMITED NUMBER OF TISSUE TYPES TO DEAL WITH THROUGHOUT DEVELOPMENT. YOU HAVE THIS FOREBRAIN EPITHELIUM, WE HAVE THE INTERVENING FRONTAL NASAL MESENCHYME AND THEN YOU HAVE THE SURFACE ECTO DETERMINE OF THE HEAD PART OF WHICH BECOMES NEURALLIZED AND GIVES RISE TO THE OLFACTORY EPITHELIUM. EVENTUALLY OF COURSE THIS FOREBRAIN THEME IS GOING TO GENERATE A HUGE VARIETY OF CELLS AND THEY WILL GIVE RISE TO ALL OF THE NEURONS THAT SUBSTITUTE THE FOREBRAIN AND MAKE THE CIRCUITS THAT DO THE HEAVY LIFTING OF BEHAVIORS. JUST JUST PHRASE THE QUESTION HOW IS THE STEM CELL IDENTITY IN THESE TWO TISSUES BASICALLY BEGIN THEIR EXISTENCE ADJACENT TO EACH OTHER, SEPARATED BY THIS MESENCHYME WHICH I'LL TELL YOU NOW AND DISCUSS THIS LATER DERIVED PRIMARILY FROM THE CRUST, HOW IS STEM CELL IDENTITY ESTABLISHED IN BOTH TISSUES, WHAT ARE THE COMMONALITIES AND WHAT ARE THE DIFFERENCES. AND OF COURSE THIS IS AN IMPORTANT QUESTION BECAUSE AS LEO MENTIONED, THESE STEM CELLS THAT ARE INITIALLY ESTABLISHED IN THIS TISSUE, SOME PROGENY OF THEM EITHER DIRECTLY OR INDIRECTLY IS GOING TO BE RETAINED IN THE TISSUE THROUGHOUT LIFE TO GENERATE OLFACTORY RECEPTOR NEURONS OVER THE COURSE OF A LIFETIME. AND IT'S OF COURSE IT'S THOUGHT THAT THIS PROCESS IS ONE OF THE FIRST THAT'S COMPROMISED IN A VARIETY OF DEGENERATIVE DISORDERS. AS I SAID, THE STEM CELLS HERE IN THE FOREBRAIN HAVE TO GIVE RISE TO CORTICO AND SUBCORTICO NEURONS THAT ARE REALLY ESSENTIAL FOR ANYTHING THAT DEFINES ANY ORGANISM THAT HAS TO BEHAVE IN THE ENVIRONMENT. SO IN THIS SENSE, THIS QUESTION OF STEM CELL IDENTITY REALLY BECOMES A SIGNIFICANT ONE AS WELL AS AN EMBRYONICALLY INTRIGUING BECAUSE OF THE PROXIMITY OF THESE TWO POPULATIONS. SO IS STEM CELL IDENTITY ESTABLISHED? WELL WE'RE GOING TO THINK ABOUT THAT IN THE FOLLOWING WAY. WE'LL LOOK AT THE OLFACTORY EPITHELIUM. YOU BASICALLY HAVE TWO TISSUES YOU CAN DEAL WITH TO INTERACT, EPITHELIUM AND MESENCHYME. IN THE FOREBRAIN YOU HAVE THE SAME TISSUES AND WE'RE GOING TO ASK THREE QUESTIONS. FIRST WE'RE GOING TO ASK WHO ARE THE STEM CELLS. I CAN TELL YOU THE ANSWER FOR THAT IS ALREADY KNOWN TO THE FOREBRAIN BUT IT WASN'T KNOWN FOR THE OLFACTORY EPITHELIUM SO WE'RE GOING TO SPEND A LITTLE TIME DISCUSSION YOU HOW WE GOT THAT ANSWER. WHAT IS THE SOURCE OF KEY SIGNALS AND WHAT ARE THE SIGNALS. SO THOSE ARE THE THING WE'RE GOING TO TAKE APART TODAY AND WE'RE GOING TO SEE HOW IN TOTALITY THAT GIVES US A PICTURE OF HOW YOU ACTUALLY DIRECT THESE SIMPLE NEUROPATHY TO ACQUIRE THE INSTRUCTIONS NECESSARY TO GIVE THE STEM CELLS THE CAPACITY TO THEN MAKE THEIR PROGENY IN APPROPRIATE NUMBERS AT APPROPRIATE PLACES WITH THE APPROPRIATE IDENTITY. WE KNOW THE IDENTITY FOR THE STEM CELLS OF THE FOREBRAIN AND THAT MAKES OUR JOB EASIER IN TERMS OF ANSWERING THIS QUESTION BECAUSE ONE'S ALREADY BEEN ANSWERED FOR US. BUT FOR THE OLFACTORY EPITHELIUM, SURPRISINGLY AS LATE AS 2010, THIS QUESTION WAS NOT ANSWERED. SO IN THE EARLY PART OF THE FIRST DECADE OF IN NEW MILLENNIUM, I AND SEVERAL OF MY COLLEAGUES SET OUT TO ANSWER THIS QUESTION IN MY LABORATORY. SO WHAT ARE THE STEM CELLS. WE'RE GOING TO THINK ABOUT STEM CELL IDENTITY IN THREE VERY SIMPLIFIED STEPS. FIRST OF ALL YOU IDENTIFY A FATE BIASED CELL, A PRECURSOR STEM CELL. IT'S GOING TO HAVE SOME PROPERTY. IT'S GOING TO HAVE SLOW AND SYMMETRIC POSITION. THERE'S GOING TO BE ANOTHER POPULATION OF PRECURSORS, TRANSIT AMPLIFYING CELLS THAT ARE GOING TO HAVE THE CAPACITY TO EXPAND THE POPULATION ONCE THE FATE BIAS STEM CELLS DIVISION OF SLOW AND SYMMETRIC IS ESTABLISHED. AND THIS PROGENY OF THE PRECURSOR STEM CELL, THIS TRANSIT AMPLIFYING CELL IS GOING TO HAVE FASTER CELL CYCLE KINETICS. IT'S GOING TO DIVIDE ASYMMETRICALLY AND IT'S GOING TO BE THE CELL THAT PRIMARILY GIVES RISE TO THE DIFFERENTIATED CELLS IN LINEAGE. AND SO TO THINK ABOUT THIS, IN TERMS OF MOLECULAR TOOL THAT WE CAN MANIPULATE THAT ARE APPROXIMATELY ESSENTIAL AND ALSO GIVE US A WAY OF DIVIDING THESE POPULATIONS UP, WE KNOW TRANSCRIPTIONAL REGULATOR'S A KEY FOR EACH OF THESE STEPS. BUT WE ALSO KNOW THAT THERE ARE A SERIES OF SIGNALS THAT ACT SELECTIVELY FOR EACH OF THESE CELL CLASSES THAT ARE ABSOLUTELY ESSENTIAL FOR DEFINING THE FATE BIASED STEM CELLS, FOR PERMITTING THE TRANSITION TO A TRANSIT AMPLIFYING IDENTITY, FOR DRIVING THE RAPID AND ASYMMETIC POSITION AND FOR FINALLY SIGNALING THE PROGENY OF THOSE TRANSIT AMPLIFYING TO BEGIN TO ACQUIRE, TO BECOME POST MOTOTIC AND ACQUIRE THEIR IDENTITY. WE'LL TAKE APART THE OLFACTORY EPITHELIUM, WHAT ARE THE MOLECULAR PLAYERS THAT DEFINE EACH OF CELL TYPES. WE'RE GOING TO DEFINE BASED ON THE CELL CYCLE KINETIC, THE ABILITY TO PROVIDE SYMMETRICALLY OR ASYMMETRICALLY AND BY VIRTUALLY DOING THAT WE'RE GOING TO DO SOME GENETIC EXPERIMENTS THAT ACTUALLY DEFINE THE IDENTITY OF THE OLFACTORY EPITHELIAL STEM CELLS. SO THE TRANSIT AMPLIFYING CELLS. SO HOW DO WE FIND AN OE STEP CELL. IT'S SORT OF EMERGED THERE ARE RULES YOU HAVE TO USE TO DO THIS. YOU HAVE TO HAVE A UNIQUE MOLECULAR SIGNATURE. USUALLY THAT SIGNATURE IS BASED ON A SIGNATURE OF TRANSCRIPTION FACTORS. BUT THAT ALONE ISN'T SUFFICIENT FOR IDENTITY. THEY HAVE TO HAVE A UNIQUE LOCATION. THEY'RE A NICHE. THEY HAVE KINETICS WHICH I'VE OUTLINED. WHEN THE PRECURSORS ARE GONE, THE CELLS THAT ARE MADE BY THOSE STEM CELLS HAVE TO ALSO BE GONE. SO ORN, THE NEURONS WHICH ARE CHEMO SENSORY NEURONS BUT PICKUP PHEROMONES AND THE GNRH NEURONS WHERE SUZANNE WHO IS UNFORTUNATELY NOT HERE, WORKS ON, WOULD NOT BE GENERATED. THOSE ARE THESE THREE CELL TYPES. THE ORM'S VRN'S WHICH LOOK LIKE ORN'S BUT HAVE A DIFFERENT FUNCTION AND OF COURSE THE GENERATION WHICH LEAD THE OPEN THEME VERY EARLY AND MIGRATE INTO THE FOREBRAIN AND ARE ABSOLUTELY ESSENTIAL FOR REPRODUCTION. FINALLY THERE HAS TO BE REGULATORY RELATIONSHIPS BETWEEN THE TRANSCRIPTION FACTORS THAT DEFINE EACH OF THE SUBTYPES OF CURSOR CELLS, THE STEM CELLS AND THIS IS REFERRED TO SOMETIMES TRANSCRIPTIONAL CODE. SO WE'RE GOING TO SEE WHETHER OR NOT WE CAN FIND CELLS. AND SATISFY EACH OF THESE CRITERIA THAT ARE THERE FOR IDENTIFIED AS THE OLFACTORY EPITHELIUM NERVES IN THE NEUROSTEM CELL. SO DISTINCT PRECURSOR DOMAINS. WHAT WE FOUND BY BASICALLY ROUNDING UP THE USUAL SUSPECTS AND LOCALIZING THEM IN THIS VERY SIMPLE OLFACTORY EPITHELIUM THAT 11.5 IN EMBRYO GENESIS. IT LOOKS LIKE A MITTEN. THIS IS THE LATERAL SIDE, THIS IS THE MEDIAL SIDE. THIS IS DORSAL, THIS IS VENTRAL. THE MITTEN IS WHERE THE -- NEURONS EMERGE. THIS IS WHERE MOST OF THE GENERATIONAL NEURONS COME FROM. WHAT YOU SEE IS THAT IN TERMS OF A NUMBER OF TRANSCRIPTIONAL REGULATORS, SOX2 WHICH ISIAN TO ALL NEUROSTEM CELLS DEPENDING ON THE DOSE SUBJECT. SCL1 KNOWN AS MASH 1 WHICH IS A BCHL CELL HAS AN IMPORTANT FOR MAKING THE OLFACTORY EPITHELIUM. THE AIS ONE IS INVOLVED IN MATT POSTS HAVE DIFFERENTIAL DISTRIBUTION. I WANT YOU TO NOTICE THIS LATERAL REGION THAT WRAPS UP IN CONVENTIONAL REGION VERSUS THIS MEDIAL REGION. THE LATERAL IDENTIFIED BY -- AND PBX. THIS MEDIAL AND DORSAL REGION IDENTIFIED BY AFCL1. AND ALSO I WANT YOU TO NOTICE THIS RADIANT OF SOX 2 LOW LATERAL HIGH MEDIAL BECAUSE IT TURNS OUT THAT THOSE GRADIENTS GAVE US THE FIRST HINT OF CELLULAR IDENTITY WITH LATERAL CELLS VERY DISTINCT, AND APPARENTLY AS THE SOX 2 DOSAGE INCREASES, THE EXPRESSION OF MIAS DECLINES AND SIMILARLY WITH ASCLI AS -- DESILENT SACL1 INCREASES. THIS SUGGESTS THERE MIGHT BE SOME RELATIONSHIP BETWEEN PROGENITOR CELLS THAT ARE HERE IN TERMS OF THEIR EXPRESSION OF KEY DETERMINANTS AND PROGENITOR CELLS THAT ARE HERE. AND FURTHERMORE WHEN WE LOOK AT SOME OTHER TRANSCRIPTION FACTORS, WE SEE CONVERSE GRADIENTS. IN THIS CASE, SIX ONE WHICH IS THOUGHT TO BE IMPORTANT FOR OLFACTORY EPITHELIUM OR PROGENESIS. YOU SEE ITS GRADIENT IS HIGH IN THE LATERAL AND DORSAL AND LOW IN THIS MEDIAL AND VENTRAL REGION. SO FROM THESE SORTS OF ANALYSIS. WHAT WE DETERMINED WAS THERE WAS A NESTED PATTERN OF EITHER GRADED OR FAIRLY DISTINCT BOUNDED TRANSCRIPTION FACTORS THAT ACTUALLY DIVIDED THE OLFACTORY EPITHELIUM EARLY INTO TWO REGIONS. THIS LATERAL REGION AND THIS MEDIAL REGION. AND THE QUESTION WAS WAS WHETHER OR NOT THERE WAS SOME RELATIONSHIP BETWEEN THE PROLIFERATIVE AND DIVISION CHARACTERISTICS OF THESE CELLS IN THE LATERAL REGION VERSUS THE MEDIAL CELLS THAT WOULD GIVE US A SENSE OF WHETHER OR NOT WE COULD LOCATE WHERE STEM AND TRANSIT AMPLIFYING CELLS WERE. NOW THE FIRST THING WE DID WAS WE MEASURED THE CELL CYCLE OF THESE CELLS IN VIVO. AND IN ORDER TO DO THAT, WE USE A TRICK USING TWO DIFFERENT ANALOGS, BRDU AND IDU. YOU INJECT THEM AT SLIGHTLY DIFFERENT TIMES AND YOU USE THE PROPORTION OF DOUBLY LABELED VERSUS SINGLY LABELED CELLS AS A TOOL TO ACTUALLY ESTIMATE BOTH THE LENGTH OF S PHASE AND THE OVERALL CELL CYCLE TIME. THE WAY THAT WE DID THIS WAS WE DIVIDED UP THE OLFACTORY EPITHELIUM IN ITS ENTIRE ANTERIOR TO POSTERIOR DOMAIN INTO TEN EQUIVALENT SECTIONS. THEN WE DID THE CELL TIMES TO DETERMINE CELL CYCLE TIMES AT EACH OF THESE DOMAINS. WHAT YOU SEE IS THAT THE LATERAL DOMAINS WHICH IS SECTOR ONE, TWO, THREE AND FOUR. SO IF WE GO BACK, YOU'LL SEE ONE, TWO, THREE, FOUR. AND THIS VENTRAL REGION HERE. WITH THOSE REGIONS HAVE MUCH LONGER CELL CYCLE TIMES THAN THE MEDIAL AND DORSAL DOMAINS. I WILL TELL YOU NOW THAT THIS IS WHERE NEUROGENESIS HAPPENS, THIS IS WHERE THE MATURING NEURONS ARE. THESE CELLS ARE NOT KNOWN FOR THE MOST PART BUT THEY DO EXPRESS NEURON MARKERS. SO THESE CELLS HAVE A LONGER CELL CYCLE TIME. IN TERMS OF WHETHER WE'RE LOOKING FOR, THEY'RE GOOD CANDIDATES BECAUSE WE SAID THE STEM CELL IS GOING TO DIVIDE SLOWLY. AND INDEED, THESE SECTORS ARE EXACTLY THE SECTORS WHERE MAI S&P BX ARE HIGH. SOX 2 IS LOW AND THESE SECTORS ARE "OR -- IS HIGH AND SOX 2 RADIANCE INCLIMBS. WHAT WE SEE TO CONFIRM THIS IS A PH3 AND WE'LL DO THE SAME MAPPING WITH THE SAME SECTORS, ONE, TWO, THREE, FOUR, FIVE, ETCETERA. AND WHAT WE SEE WHEN WE DO THAT IS THE MITOTIC ACTIVITY OF THE CELLS IN SECTORS ONE, TWO, THREE AND FOUR IS VERY LOW. AND IS SIGNIFICANTLY AND DRAMATICALLY INCREASED IN THESE SECTORS. SO WE KNOW THAT THESE CELLS ARE DIVIDING MORE RAPIDLY. THEY HAVE CELL CYCLE, TIME IS SHORTER. THESE ARE DIVIDED MUCH MORE SLOWLY IN THEIR CELL CYCLE TIME IS LONGER. THESE MAKE THESE CELLS REALLY GOOD CANDIDATES FOR THE STEM CELLS. HOW ARE WE GOING TO WORK OUT WHETHER OR NOT THEY ARE INDEED THAT OR NOT. SO ONE THING WE DID WHICH WAS AN EASY BUT IT'S ACTUALLY A CRITERIA THAT'S OFTEN USED TO DEFY STEM CELLS IS WHETHER OR NOT THEY WERE SO-CALLED LABEL-RETAINING. BASICALLY WHAT YOU DO IS LABEL DNA DURING S PHASE AND YOU SEE WHETHER OR NOT AFTER A LONG INTERVAL, IN THIS CASE -- WHO ARE THE CELL THAT ACTUALLY RETAIN HEAVILY THE DNA LABEL AND THEIR NUCLEI. WHAT WE FOUND IS THAT THE ONLY CELLS THAT WE COULD SEE THAT WERE HEAVILY LABELED WITH -- THAT WE'RE STILL DIVIDING THESE BASAL PROGENITORS NOW, MOSTLY IN THE LATERAL REGIONS THAT EXPRESSION HIGH LEVELS OF MEIS1 AND LOWER LEVELS OF SOX 2. THAL CELLS THAT WE SAW LABELED, WE NEVER SAW BRD LABELED CELL THAT WAS ACTUALLY LABELED WITH MASH OR ASCL1 NOR DID WE SEE ANY HIGH SOCKS LABELED BUT WE DID SEE SEVERAL MATURING OMP EXPRESSING NEURON IN THE MEDIAL PART OF THE EPITHELIUM THAT WERE LABELED. SO WHAT THIS SUGGEST IS THAT THESE CELLS ARE DIVIDING VERY SLOWLY AND THEY ARE RETAINED IN THE TISSUE POTENTIALLY AS SLOWLY DESCRIBING STEM CELLS. THE NEXT THING WE ASK IS WHETHER OR NOT THEY HAD DISTINCT CELLS OF MODE DIVISION. WE'RE GOING TO MICRO DEFECT THIS TISSUE OUT, ARE WE'RE GOING TO CLEAN IT UP AND DISASSOCIATE THE LATERAL FROM THE MEDIAL. WE'LL DO IT FROM RAID YENT CELLS AND WE'LL ACTUALLY LOOK AT INDIVIDUAL PAIRS OF CELLS AND ASK A HOW THEY DIVIDE AND B WHETHER OR NOT THEY SYMMETRICALLY OR ASYMMETRICALLY DISTRIBUTE DETERMINANCE. WHETHER THEY RETAIN THE PRECURSOR IDENTITY BASED ON THE EXPRESSION OF MEIS1, SOX 2 OR THEY ARE EITHER SYMMETRICALLY TERMINALLY NEUROGENIC OR ASYMMETRICALLY TERMINALLY NEUROGENIC. THE CELLS YOU HAVE IN THE OLFACTORY EPITHELIUM WHERE THE CELL CYCLE TIMES ARE LOW AND WHERE YOU HAVE LABEL RETAINING CELLS IS THAT YOU MAKE SYMMETRICALLY DIVIDING PRECURSOR PRECURSOR PAIRS. IT'S EXACTLY THE OPPOSITE PICTURE IN THE MEDIAL OE, YOU MAKE SYMMETRICALLY DIVIDING NEURON NEURON PAIRS. THIS GIVES US ANOTHER PIECE OF EVIDENCE THAT THE STEM CELLS MUST BE HERE IN THE LATERAL OE. NOW THE NEXT THING WE'RE GOING TO DO IS WE'RE GOING TO BEGIN TO TAKE THIS APART GENETICALLY. I TOLD YOU ASCO1 OR MASH 1 IS GETTING THE RIGHT NUMBERS OF ORM. THIS IS JOAN JET -- SHOWN GENETICALLY IN THE DMO AND ITS COLLEAGUES BECAUSE MUTANTS DISRUPT OLFACTORY EPITHELIAL DEVELOPMENT BUT THEY DIDN'T KNOW HOW. SO WHAT WE THOUGHT WAS THAT IT WAS VERY POSSIBLE AFCL1 EXPRESSING PROGENITORS WERE ACTUALLY NECESSARY FOR SPECIFYING ALL OF THE CELL CLASSES BUT RATHER FOR EXPANDING THEIR NUMBERS BY VIRTUE OF ASYMMETIC AND TURNAL NEUROGENIC DIVISIONS. SO WHAT WE DID IS LOOKED AT AN ASCL1 MUTANT AND WE SIMPLY ASKED WHETHER OR NOT YOU COULD RECOVER OMP EXPRESSING NEURONS GENERATED EARLY AT THIS EARLY TIME VERSUS ONE DAY LATER. SO WE'RE BIRTH DATING CELLS WITH ONE DAY INTERVAL. WHAT YOU SEE IS THE INITIAL COMPLEMENT OF OMP EXPRESSING CELL IS INDISTINGUISHABLE IN THE WILD TYPE AND THE MUTANT. HOWEVER, WITHIN A DAY YOU HAVE ALMOST NO KNOWN P GENERATED CELLS BEING MADE. NOW WHAT WE DO NOW HERE IS THAT THE OTHER, SO IT'S NOT NECESSARY FOR INITIAL GENESIS OF ORNS OR THE VRNS BUT IT IS NECESSARY FOR THE EXPANSION OF THE POPULATION. INDEED EVEN THE GENERATION MEURNLZ ARE MADE IN THIS MOO -- NEURONS ARE MADE IN THIS MUTE UN. -- IT DOES SEEM AFTER YOU SPECIFY THE STEM CELLS NOT DEPENDENT ON ASCL1 FOR THEIR IDENTITY BUT ALL OF ITS DOWN STREAM TARGETS ARE ESSENTIAL FOR EXPANDING THE POPULATION PARTICULARLY FOR THE ORN'S. THAT PUTS ASCL1 IN A VERY DIFFERENT PLACE THAN INITIALLY THOUGHT TO BE. NOW, WE FIND A UNIQUE PRECURSOR CLASS BUT THE QUESTION IS WHAT ARE THESE PRECURSOR CLASS ACTUALLY FOUND AND WHAT ARE THEIR DYNAMICS. UNTIL YOU ANSWER THAT QUESTION, WE BEGUN USING AN ASCL1 PRECONDITIONED ALLELE THAT IS ACTION CAUSING RECOMBINATION OF JUST A REPORTER JUST TO SEE INITIALLY WHAT HAPPENS TO DISTINCT COHORTS OF CELLS THAT ARE PASSING THROUGH AN ASCL1 EXPRESSING NODE, IN THE OLFACTORY EPITHELIUM, AND SEE WHO THEIR IDENTITY IS. CAN YOU DEFINE DISTINCT CLASSES OF PRECURSORS BASED ON THEIR PASSING THROUGH A PARTICULAR NODE, PARTICULARLY DISTINCT CLASSES WHAT WE BELIEVE ARE THE AMPLIFYING CELLS THAT ARE ACTUALLY MAKING THE POPULATIONS OF NEURONS. SO WHAT WE SAW WHEN WE DID THIS IS WE INJECTED TAMOXIFEN AND WE SAW THAT THE CELLS THAT HAD PASSED THROUGH AN ASCL1 EXPRESSING NODE THEIR PROGENY ARE BASICALLY ACCUMULATED IN THESE REGIONS OF THE DORSAL AND MEDIAL OLFACTORY EPITHELIUM. AND SO IF WE ANALYZE THIS BY SECTORS, WHAT WE SEE IS YES INDEED THEIR FREQUENCY IS HIGHER. AND WE BEGUN TO LOOK AT JUST OTHER MARKERS THAT ARE RELATED TO THIS BUT WE FIRST SEE IS THAT THIS IS ACTUALLY THE REGION WHERE YOU SEE THE HIGHEST INCORPORATION OF BRD. THIS IS MITOTICALLY ACTIVE DNA SYNTHESIS ACTIVE REGION. THE CELL CYCLE TIMES ARE SHORT. SO THIS IS AN ACTIVE REGION OF CELL PROLIFERATION. AND FURTHERMORE, WHAT'S INTERESTING THOUGH IS THE ASCL1 CELLS ARE NOT DOUBLE LABELED. WHEN WE LOOK AT WHERE NEURONS ARE DIFFERENTIATING IN TERMS OF THEIR CAPACITY TO EXTEND AXONS, WE SEE THIS IS NOT A REGION WHERE NERVE CELLS THAT ARE ACTUALLY ACTIVELY EXTENDING AXONS AND BEGINNING TO GO THROUGH THE NEXT STEPS OF DIFFERENTIATION. WHAT WE THINK THIS DEFINES, THIS DEFINES A DISTINCT REGION OF TRANSIT AMPLIFYING CELLS THAT ARE ACTUALLY GENERATING BY TERMINAL NEUROGENIC POSITIONS AS WELL AS ASYMMETIC DIVISIONS. THESE NEURAL BLASTS THAT ARE THEN GOING TO BEGIN TO DIFFERENTIATE. WHAT YOU HAVE IN A SENSE AND I'M GOING TO SHOW YOU THIS IN A MOMENT NOW, A MECHANISM FOR MULTIGENESIS OF -- BECAUSE IF WE TAKE THE OE APART IN THIS WAY, NOW WE'RE GOING TO STRETCH IT OUT AND WE'RE GOING TO INDICATE HOW THE VARIOUS PUNITIVE STEM CELLS VERSUS TRANSIT AMPLIFYING CELLS ARE RELATED. IF YOU IMAGINE THAT AT THIS BOUNDARY AND AT THIS BOUNDARY AS WELL, THE STEM CELLS CAN GENERATE TRANSIT AMPLIFYING PROGENITORS THAT CAN THEN ASYMMETRICALLY DIVIDE AND GENERATE MORE TRANSIT AMPLIFYING PROGENITORS. THEN IF YOU IMAGINE THEY CELLS BEGIN TO DIFFERENTIATE AS NEURONS. WHAT YOU HAVE IS YOU HAVE A WAY OF ESTABLISHING A PROGRESS ZONE, IF YOU WILL THAT THEN FILLS IN WITH NEUROGENESIS. IF YOU THINK ABOUT THIS ITERATIVELY, WHAT THAT COULD POTENTIALLY DO IS GIVE YOU A WAY OF BUILDING THESE TERMANCE THAT HAVE TO PROTRUDE BY ESTABLISHING A NUMBER OF PROGRESS ZONES AT THE BORDERS OF WHERE THE STEM CELLS ARE FOUND VERSUS WHERE THERE ARE SIGNALS THAT ARE APPROPRIATE FOR DRIVING THE TRANSIT AMPLIFYING CELLS. SO THE NEXT QUESTION WE'RE GOING TO ASK IS, ARE IS THIS KIND OF A MODEL KEPT WITH THE TRANSCRIPTIONAL REGULATORY RELATIONSHIPS THAT WE CAN DISCERN BETWEEN THESE FACTORS THAT AT LEAST MARK THESE DISTINCT POPULATIONS OF CELLS. AND IS THIS CONSISTENT WITH THE DISTINCT LOCAL SYNAPSE THAT WE KNOW MAY ACT IN THE OLFACTORY EPITHELIUM IN INFLUENCE NEUROGENESIS AND DIFFERENTIATION. SO THE FIRST QUESTION THAT I'M GOING TO ASK IS WHETHER OR NOT SOX 2AEIS1 AND ASCL1 IS PART OF A TRANSCRIPTIONAL CODE. IN ORDER TO DO THIS WE TOOK A SLIGHTLY DIFFERENT APPROACH INITIALLY AND THAT IS THAT WE SET UP AN ELECTROPREPARATION WHERE WE TAKE THE LATERAL OR MEDIAL OLFACTORY EPITHELIUM AND BASICALLY EXPRESS IN AN AUTONOMOUS WAY ONE OR THE OTHER OF THESE FACTORS AND SEE WHAT THE CONSEQUENCES WERE FOR EXPRESSION OF DOWN STREAM POTENTIAL GENES AND NEUROGENESIS. SO WE'RE GOING TO FOCUS ON DOING THAT IN THE LATERAL OLFACTORY EPITHELIUM AND WE'RE GOING TO ANALYZE CELLS IN THIS TRANSITION ZONE WHERE NORMALLY THE ONE GROUP OF CELLS ARE FOUND AND WHERE THE ASCL1 CELLS AS WELL AS NEWLY GENERATED NEURON ARE FOUND AT THAT BOUNDARY. WHAT WE SEE WHEN WE DO THIS, THIS IS A CONTROLLED GFP INJECTED SINGLE CELL BLUE AS MEIS1 AND YOU SEE THAT THEY CO-LOCALIZE. WHEN WE OVER EXPRESS SOX IN A FIELD OF CELLS THAT NORMALLY EXPRESS MEIS1 AND HIGH LEVELS, WHAT WE SEE IS WE SEE VERY VERY RARELY THAT MEIS1 CONTINUES TO BE EXPRESSED. AND THIS IS THE QUANTIFICATION. SO THIS IMPLIES THAT DOSAGE OF SOX 2 IS A KEY REGULATOR OF THE EXPRESSION OF MEIS ONE. SO THESE CELLS THAT ARE SLOWLY DIVIDING, EXPRESSING MEIS1, THEIR EXPRESSION OF MEIS1 IS REGULATED BY THAT CONVERSE GRADIENT OF SOX 2. AND SIMILARLY WHEN WE DO THE SAME EXPERIENCE, WE LOOK AT THE EXPRESSION OF ASCL1, WE SEE THAT ASCL1 IS RARELY EXPRESSED WHEN WE JUST DO THE CONTROL. ALSO WE CAN DO THIS WITH MEIS1 WHICH I'LL SHOW YOU IN A MOMENT. BUT WHENEVER WE OVER EXPRESSION SOX 2 AT HIGH LEVELS IT'S THAT DOSAGE OF SOX 2 THAT ACTUALLY COMPLETELY REVERSES THE IDENTITY AND ACTUALLY CAUSES ASCL1 TO BE EXPRESSED IN ALMOST EVERY CELL THAT SOX 2 IS EXPRESSIONED AT HIGH LEVELS AND NORMALLY WOULD NOT, THESE CELLS WOULD NOT BE EXPRESSING THE SCL1. THIS IMPLIES THAT THE SOCKS 2 GRADIENT IS ACTUALLY KEY FOR REGULATING EITHER THE IDENTIFY SLOWLY DIVIDING THE PUNITIVE STEM CELLS VERSUS THE MORE RAPIDLY PROVIDED ASCL1 EXPRESSING TRANSIT AMPLIFYING CELLS. AND SIMILARLY WHEN WE DO THAT EXPERIMENT BY RAISING SOX 2 AND ASCL1 WE INCREASE THE LEVEL OF NEUROGENESIS. THESE CELLS LOOK LIKE ORM, THEY HAVE A SINGLE AXON AND DENDRITIC PROCESSES AND YOU SEE THE NUMBER OF NEURONS IN THIS REGION INCREASES DRAMATICALLY. SO THIS IS CONSISTENT WITH A VERY SIMPLE TRANSCRIPTIONAL REGULATORY MECHANISM WHERE SOX 2 DOSAGE IN GRADED FASHION REGULATED BY SIGNALS THAT WE'LL DISCUSS IN A MOMENT IS ACTUALLY KEY FOR EITHER RETAINING MEIS1 EXPRESSION AND ONE IMAGINES THE IDENTITY OF STEM CELLS TO GENERATE THE LINEAGE VERSUS FOR PUSHING THOSE CELLS INTO A SUBPOPULATION THAT IS TRANSIT AMPLIFYING AND CAN EXPAND OUT THE POPULATION OF NEURONS. NOW WE'RE GOING TO DO THE EXACT OPPOSITE EXPERIMENT. WE'RE GOING TO DO THIS WITH MEIS1. EITHER ALONE OR WITH ITS PBX COFACTOR. WHAT WE SEE IS THERE'S NOT A RELIANCE OF PBX COFACTOR BINDING WITH MEIS1 WHICH IMPLIES THAT THE MEIS1 DNA BINDING DOMAIN IS KEY FOR THIS. WE ACTUALLY NOW MAKING MUTANTS, WE'RE MUTATING THAT DOMAIN AS WELL AS SEVERAL OTHERS. BUT I DON'T HAVE ANSWERS YET. AND WHAT YOU SEE IS THAT BY VIRTUE OF DOING THIS, YOU DIMINISH THE EXPRESSION OF ASCL1 DRAMATICALLY. SO THIS IMPLIES THAT THERE'S ACTUALLY CROSS REGULATION THAT MEIS ONE IS NORMALLY SUPPRESSING ASCL1 WHICH IS IMPORTANT FOR THE TRANSIT AMPLIFYING TO STAND OUT THE NUMBERS OF CELLS. AND SO THEREFORE YOU HAVE SORT OF A SERVO MECHANISM, IF YOU WILL WHERE YOU HAVE A SOX 2 GRADIENT WHICH IS CO-SO THE CONTROL OF THIS GRADIENT IS GOING TO BECOME KEY. AT THE TOP OF THE GRADIENT ASCL1 EXPRESSION IS FAVORED. AT THE BOTTOM OF THE GRADIENT MEIS1 EXPRESSION IS FAVOREDDED. MEIS1 EXPRESSES ASCL1 AND BY VIRTUE OF THIS YOU REMAIN THE IDENTITY OF THESE CELLS AS SLOWLY DIVIDING SYMMETRICALLY DIVIDING STEM CELLS SO WE'RE GOING TO TEST THAT. THE FIRST THING WE'RE GOING TO DO IS ASK WHETHER OR NOT COX 2 LEVELS GENETICALLY IF WE MANIPULATE GENETICALLY CAN REGULATE MEIS ONE, PROLIFERATION AND NEUROGENESIS. SO WHAT I'M GOING TO SHOW YOU NOW IS THIS IS THE NORMAL CASE AND THEN I'M GOING TO USE A SOX 2 HYPER MORPH AND THIS IS WHERE THE MEIS1 EXPRESSING CELLS ARE IN THIS GRADED FASHION. NOW WE'RE GOING TO LOWER SOX 2 EXPRESSION SO THAT IT'S UNIFORM ACROSS THE ENTIRE EPITHELIUM AND IT'S QUITE LOW. IT'S BY A 20% LEVELS IS WHAT IT NORMALLY WOULD BE. WE'RE GOING TO LOOK AT WHAT HAPPENS. WHAT HAPPENS IS ALL OF THESE CELLS NOW EXPRESS MEIS1 ALL THE WAY ACROSS THE BOARD. NOW, NONE OF THEM EXPRESS ASCL1 AND NASH 1 ALTHOUGH NASH 1 IS EXPRESSED IN THE FOREBRAIN JUST FINE. WHAT HAPPENS ALSO IS THAT YOU SLOW DOWN THE CELL CYCLE KINETIC. THESE CELLS ARE NOW, THEY'RE ALL IN THE IS1 EXPRESSION AND DIVIDED MORE SLOWLY. THIS IS PH3 THAT WITH DO THE SAME THING WITH THE CELL CYCLE TIMES. FINALLY WHAT HAPPENS IS THAT, I'LL SHOW YOU THIS HERE. THE NUMBER OF ORM DECLINES DRAMATICALLY BEYOU STILL MAKE ORM'S BRM'S AND GENERATION RUNS. SO WHAT THIS IMPLIES IS THAT ALTHOUGH SOX 2 DOSAGE IS ABSOLUTELY NECESSARY FOR CONTROLLING THE IDENTITY OF THE STEM FROM THE TRANSIT AMPLIFYING CELLS. IT'S NOT ABSOLUTELY NECESSARY FOR ALLOWING SOME LEVEL OF NEUROGENIC TERMINAL DIFFERENCE -- DIVISION AND APPROPRIATE DIFFERENTIATION OF ALL THE MAJOR CELL CLASSES. SO WHAT THAT TELLS US IS WE HAVE A CELL TYPE THAT IS LIKELY THE STEM CELL AND THAT THE CONTROL OF SOX 2 LEVELS IS ACTUALLY KEY FOR THE ADVANCEMENT IN TERMS OF PROLIFERATIVE CAPACITY FROM THE STEM CELL IDENTITY TO THE TRANSIT AMPLIFYING IDENTITY TO MAKE THE TISSUE. NOW THE NEXT QUESTION THAT ARISES IS WHAT IS THE SOURCE OF KEY SIGNALS THAT MIGHT REGULATE THAT PROCESS? AND WHEN WE LOOK AT WHAT'S OPPOSED TO THIS PRESUMPTIVE OLFACTORY EPITHELIUM OR EVEN THE OPTIC VESICLE BACK HERE, WHAT WE SEE IS THE ONLY OTHER INTERVENING TISSUE IS THIS FRONT NASAL MESENCHYME -- SO THIS IS WHAT WE HAVE TO DEAL WITH. AT E9 WHAT WE SEE IS WE BASICALLY HAVE THREE ISSUES THE FRONTAL MESENCHYME, THE OLFACTORY EPITHELIUM, THE FOREBRAIN EPITHELIUM. IT DIFFERENTIATES SLIGHTLY AND THEN WE HAVE THIS INDENTATION OF THE DEVELOPING OLFACTORY EPITHELIUM BY E11. NOW WHAT WE'RE GOING TO ASK IS WHETHER OR NOT ME INTERACTIONS ACTUALLY SPECIFY THESE STEM CELLS. WE KNOW FROM WORK DONE PREVIOUSLY IN MY LABORATORY AND OTHERS, WITH THESE INTERACTIONS ARE NECESSARY IN ORDER TO GET AN OLFACTORY EPITHELIUM. WHAT WE'RE GOING TO ASK IS WHETHER OR NOT THESE INTERACTIONS ARE KEY. WHAT WE KNOW IS ANOTHER PLACE WHERE THIS KIND OF MESENCHYME INTERACTION EPITHELIUM IS IN THE WIND AND MEIS1 IS ABSOLUTELY CRITICAL IN THE WIND BUT MESENCHYME FOR MEDIATING PROGENITOR AND STEM CELL IDENTITY AND THE SPECIFICITY OF LOCAL SIGNALING. IT'S REVERSE IN TERMS OF ITS TISSUE EXPRESSION BUT NEVERTHELESS WE HAVE A RELATIONSHIP BETWEEN MESENCHYME AND EPITHELIUM AND MEIS1 EXPRESSION. NOW THERE'S A CLASSICAL EXPERIMENT DONE BY -- I DON'T REMEMBER WHO DID IT BUT HE TRANSFERRED THE OLFACTORY PRY MORE ANNUAL -- PRY MORE ANNUAL ARE LIKELY TO BE VERY SIMILAR TO WHAT'S SEEN IN THE WIND. THE FIRST THING I'M GOING TO DO IN ORDER TO TEST WHETHER OR NOT THE SPECIFICATION OF THOSE HIGH ME SIVMENT OF IS1 LOW SOX 2 EXPRESSING CELLS ARE NECESSARY TO MAKE AN OLFACTORY EPITHELIUM WITH THE APPROPRIATE STEM CELLS AND PROGENY IS THAT I'M GOING TO TAKE THE NORMAL -- I CAN SEE WHETHER OR NOT I'LL ACTUALLY SPECIFY THE SAME COMPONENTS OF CELLS. WHEN WE DO THIS IN VITRO THE NEURONS ARE MADE LATERALLY, THEY'RE LABELED AND THEY ACTUALLY MAKE AN OLFACTORY NERVE AND I'LL SHOW YOU THIS AGAIN IN A MOMENT. ACTUALLY THE WIND MESENCHYME WITH NEUTRALIZE PRESUMPTIVE OLFACTORY EPITHELIUM. THIS MEANS THAT IN THE MESENCHYME ARE THE SIGNALS THAT ARE NECESSARY TO MAKE NEURONS BUT ARE THEY THE RIGHT NEURONS AND THAT'S WHAT WE'RE GOING TO LOOK AT. WE KNOW THESE CELLS CAN ACTUALLY EXPRESS AFCL1 OR NASH ONE AS WELL AS NEUROGENESIS WHICH IS THOUGHT TO BE DOWN STREAM TARGET. IT'S UNLIKELY IF THERE IS SPECIFICITY THESE MAY BE INVOLVED OR IF THERE ISN'T IT'S UNLIKELY THEIR INDUCTION ALONE IS LIKELY TO EXPLAIN WHY YOU GET CELL IDENTITY. SO WHAT WE SEE WHEN WE DO THIS IS THAT EVEN THOUGH THE MESENCHYME WITH NEURALLIZE THE FOAL FACTORY PLATFORM -- ACTUALLY THE MESENCHYME DOES THAT JUST FINE, I'LL SHOW YOU THAT IN A MINUTE. ALSO IT DOES NOT DIRECT EXPRESSION OF MEIS1 TO THE OLFACTORY EPITHELIUM BUT ACTUALLY RETAINS MEIS ONE EXPRESSION IN THE MESENCHYME. WHAT WE HAVE HERE IS WE HAVE IN VITRO A SYSTEM WHERE WE CAN ACTUALLY ASK WHETHER OR NOT THE STEM CELLS, THE NEURAL PROGENITORS THAT ARE HERE CAN ACTUALLY MAKE ORM OR VRM GENERATION RUNS. BECAUSE THE PRECURSORS ARE GONE. WE'VE USED AN EMBRYONIC APPROACH TO ACTUALLY ELIMINATE THESE PRECURSORS. WHAT WE'RE GOING TO DO IS LIVE WE CAN ACTUALLY IMAGE THE DIFFERENTIATING NEURONS USING THE NEUROGENESIS, WE CAN SEE THEM, WE CAN RECORD FROM THEM. AND WE CAN COMPARE THAT WITH THE CELLS THAT WE GET FROM THIS INDUCTION WHICH ARE NEURONS BUT THEY DON'T HAVE A DENDRITE -- THEY DON'T MAKE A COHERENT NERVE. IN FACT THE -- CAN'T EVEN ENTER THE MESENCHYME IN THIS HETEROLOGUS. ONE OF THE FEATURES IS THEY HAVE SODIUM CHANNEL OF A PARTICULAR TYPE THAT CAN FIRE ACTION PO POTENTIALS. THERE ARE A LOT OF THESE CELLS THAT ARE MADE WHEN YOU INDUCE EPITHELIUM WITH THE MESENCHYME WE FOUND TWO CELLS WITH THIS INDUCTION. WHAT THIS IMPLIES IS THAT THE APPROPRIATE INTERACTIONS INCLUDING HAVING THOSE SOX 2MEI CELLS ARE ABSOLUTELY NECESSARY TO MAKE THESE CELLS. THESE CELLS ARE STILL NEURONS, THEY HAVE SIMILAR CALCIUM CONDUCTED, THEY JUST DON'T HAVE THE GATED SODIUM CHANNELS. FINALLY WE SPECIFIED -- IN THIS REGION HERE IN VITRO. YOU HAVE THE RIGHT MESENCHYME INDUCING AND ACTUALLY THE FEZ2 EXPRESSION IS THE MARKER IS EXPRESSED IN THE MESENCHYME IN THE LIMB. SO WHAT THIS SUGGESTS IS THAT WHEN WE ELIMINATE THOSE SOX 2 LOW EXPRESSING MEIS HIGH EXPRESSING PROGENITORS EVEN THOUGH WE MAKE NEURONS WE DON'T MAKE THE RIGHT TYPES OF NEURONS. SO WHAT ARE THE SIGNALS THAT DO THIS FROM THE MESENCHYME OR THE EPITHELIUM? WELL THE PLAYERS IN THE LIMB ARE THE SAME PLAYERS THAT ARE AVAILABLE IN THE NOSE. THE ONLY THING IS THAT IN SOME CASES THEY'RE EITHER AVAILABLE FROM DIFFERENT SITES IN TERMS OF THE WAY THAT THEY ARE ARRANGED OR THEY ARE AVAILABLE FROM DIFFERENT TISSUES, FOR EXAMPLE SONIC EDGE. WE'RE GOING TO -- RETINAL AND BMP. ONE THING WE FOUND IS RENT NECK ACID THAT WE KNOW COMES FROM THE LATERAL BUT NOT THE MEAD YIL MESENCHYME SETS UP THE SOX 2 GRADIENT. HERE'S THE NORMAL GRADIENT ESTABLISHED IN VITRO BY HAVING THE RIGHT MESENCHYME WOULD BE FROM LATERAL TO MEDIAL YOU GET THIS GRADIENT. IF WE ACTUALLY RAISE OUR A CONCENTRATIONS, WE FLATTEN THE GRADIENT. SO THIS SUGGEST THAT RA COMING FROM THE LATERAL MESENCHYME WHICH WE KNOW IT WAS WHERE IT'S MADE IS ACTUALLY MAINTAINING SOX 2 AT LOW LEVELS OF EXPRESSION. NOW THE OTHER THINGS WE CAN SHOW THAT RA DIMINISHES THE FREQUENCY OF PRECURSOR PRECURSOR DIVISIONS THAT SUGGEST THAT ACTUALLY WHAT RA IS DOING IS ACTUALLY FURTHER SLOWING THE DIVISION OF PRECURSORS FROM THE LATERAL OE AND MAKING THERE BE LESS PAIRS. FINALLY, IF WE USE AN RA INDICATOR REPORTER TRANSGENE WHICH TELLS US WHAT CELLS ARE ACTIVATED BY RA IN THE OLFACTORY EPITHELIUM, WHAT WE SEE ARE THESE RED CELLS. AND ABSOLUTELY A HUNDRED PERCENT OF THEM DO NOT, WE CAN'T LABEL ANY OF THEM WITH BIDU. IT'S SUGGESTED THESE RA SIGNAL CELLS IN THE LATERAL EPITHELIUM ARE THE LEASE PROLIFERATIVE ACTIVE. SO THIS ACTUALLY PLACES RA AS A KEY SIGNAL FOR MAINTAINING THE IDENTITY OF THE PRESUMPTIVE STEM CELLS. THIS IS SOMETHING WE'RE ACTUALLY EVALUATING GENETICALLY, CURRENTLY. IT SEEMS TO REGULATE NEUROGENESIS VIA THEIR CONTROLLED SOX. WE'RE GOING TO DO THE SAME EXPERIENCE BUT RAISE THE CONCENTRATION OF GF8. WHAT WE SEE IS WE FLATTEN THE GRADIENT IN THE OTHER DIRECTION. WE BASICALLY FILL IN EXPRESSION OF SOX 2 SO THAT IT'S HIGH ACROSS THE MEDIAL LATERAL RANGE. AND SIMILARLY WHEN WE DO THAT EXPERIMENT ON LATERAL CELLS, WE ACTUALLY INCREASELY DOUBLE THE NUMBER OVER NEURON TO NEURON TERMINAL DIVISIONS SUGGESTING THAT FGFS ALONE IS SUFFICIENT TO MOVE A LINEAGE TO A PLACE. WE DO THIS GENETICALLY BY TAKING FTS F CONCENTRATIONS. WHAT WE SEE IS THE OPPOSITE. WE FLATTEN THE SOX 2 GRADIENT. WE LIMIT THE NUMBER OF NEURONS THAT ARE MADE. AND WHEN WE LOOK AT THE DISTRIBUTION OF ASCL1 AS WE WOULD EXPECT THE ASCL1 EXPRESSION IS LIMITED AND ALSO FREQUENCY DECLINES. SO WHAT THIS SUGGESTS TO US IS THAT FGF8 IS ACTUALLY REGULATING THE TRANSIT AMPLIFYING POPULATION BY REGULATING SOX 2 LEVELS AND INDIRECTLY AFCL1 LEVELS. SO FINALLY BMP'S ACTUALLY SEEM ALSO TO REGULATE THE ASCL1 PRECURSORS. BMP4 IS MADE AT HIGH LEVELS IN THE EPITHELIUM AND THE MEDIAL PART WHERE NEUROGENESIS IS GOING ON. WHAT YOU SEE AGAIN IS THIS PROGRESS ZONE OF PROGENITOR PROGENY WOULD PASS THROUGH AN AFCL1 NODE OF 24 HOURS EARLIER. AND WHAT WE'RE GOING TO DO IS WE'RE GOING TO LOOK AT THEIR DISTRIBUTION AND WE'RE ALSO GOING TO ASK WHERE BMP SIGNAL IS GOING ON WHERE MAPPING WHERE -- CAN BE SEEN IN THE OLFACTORY EPITHELIUM. WE'RE GOING TO SNAD ONE SIDE WHICH IS THE FOSSILS OF THE ANTIBODY FOUND THE CELLS TRANSDUCING THE SIGNALS AND BMP RECEPTOR. WE DON'T SEE MUCH DOUBLE LABELING BUT WHAT WE DO SEE IS THE DISTRIBUTION OF SNAB VERSUS THE ASCL16789 -- ALTHOUGH THEY ARE NOT DOUBLY LABELED. WHAT THIS SUGGESTS IS THERE'S A COMPARTMENT OF PROGENITORS THAT GO THROUGH AN ASCL1 EXPRESSING NODE. THOSE ARE SIGNALED BY THE BMP AND OTHERS THAT ARE ABSOLUTELY CRITICAL FOR GENERATING THE RIGHT NUMBERS OF OLFACTORY RECEPTOR NEURONS THROUGH TRANSIT AMPLIFYING. WHAT WE HAVE HERE IN THIS SIMPLE MODEL IS THAT WE HAVE THIS TRANSCRIPTIONAL CODE THAT MOVES CELLS FROM THE STEM TO THE TRANSIT AMPLIFYING IDENTITY THAT CONTROLS AT LEAST INDIRECTLY THEIR PROLIFERATION KINETICS. AND WE HAVE THESE FACTORS THAT ARE ACTING DIFFERENTIALLY EITHER ON THE STEM CELL COMPARTMENT OR ON THE TRANSIT AMPLIFYING CELLS. AND I SHOULD MENTION THAT WE KNOW THAT RA COMES FROM THE LATERAL MESENCHYME FGS8 AND BFD4 FROM THE MEDIAL EPITHELIUM. WE THINK IN SOME WAY NOW WE HAVE THE IDENTITY OF THESE CELL TYPES AND NOW WE CAN GO FURTHER WITH GENETIC AND CELL BIOLOGICAL APPROACHES TO ACTUALLY ASK HOW SPECIFICATION OF THE MATURE CELL TYPES OCCURS THROUGH THIS LINEAGE. SO, HOW ARE STEM CELL IDENTITY ESTABLISHED IN WELL FOR THE OE WE SAID THAT THE STEM CELLS ARE THESE LOW SOX 2 HIGH EXPRESSING CELLS. WE KNOW THAT'S THE STEM CELLS FOR THE FOREBRAIN. WE KNOW THE MESENCHYME IS A KEY SOURCE FOR THE SIGNALS OF THE OE. WE DON'T KNOW WHETHER SIGNALS ARE COMING FROM THROUGH SPECIFIED FOREBRAIN STEM CELLS. AND WE KNOW THAT FOR THE OE AT LEAST RAFGS AND BMP ARE KEY SIGNALS FOR VARIOUS STEPS IN LINEAGE AND WE DON'T HAVE INSIGHT FOR THIS SPECIFICATION. WHAT I'M GOING TO DO IN THESE LAST FEW MINUTES I'M GOING TO TRY TO FILL IN SOME PROVISIONAL ANSWERS TO THESE TWO QUESTIONS. FOR FOREBRAIN GLIAL DURING EARLY DEVELOPMENT. THE FIRST THING I'M GOING TO SELL YOU IS THE SIGNALING JUST LIKE THE SIGNALING THAT I SHOWED YOU THAT'S KEY FOR THE OLFACTORY EPITHELIUM IS ALSO KEY FOR SPECIFYING FOREBRAIN PROGENITORS. AND THE WAY THAT THIS IS KEY IS ACTUALLY IN A SURPRISING WAY. NOW I'M JUST SHOWING YOU HERE THAT THIS EXPLANT SYSTEM ACTUALLY WORKS AND IT RETAINS THE MARKERS ARE EARLY FOREBRAIN, EPITHELIUM EXPRESSION WHICH IS FINE IN EXPLANTS. THIS IS THE ACTUAL TISSUE FROM THE EMBRYO. SO THIS IS A GOOD SYSTEM TO ASK THESE QUESTIONS IN. AGAIN, WE HAVE THESE TISSUES NOW TO DEAL WITH. THE FOREBRAIN EPITHELIUM, THE FRONT NASAL MESENCHYME AND WE KNOW THAT THESE SIGNALS FGS8 AND SONIC HEDGE HOG COMING FROM VENTRAL AND SORT OF ANTERIOR VENTRAL REGIONS ARE ABSOLUTELY KEY FOR MAINTAINING THE INTEGRITY OF RADIAL GLIAL FOREBRAIN STEM CELLS. WHEN WE CULTURE FOREBRAIN EMTHEME ALONE, WHAT WE SEE IS THAT EVEN THOUGH WE KNOW IT RETAINS ITS MOLECULAR IDENTITY TO SOME DEGREE, SONIC HEDGE HAWK IS BARELY EXPRESSED AND THERE'S NO EXPRESSION THAT WE CAN DETECT. IF WE GIVE THAT EPITHELIUM ITS MESENCHYME WE PATTERN AND ENHANCE SONIC HEDGE HOG EXPRESSION AND WE TURN ON FGS78 EXPRESSION. SO WHAT THIS IMPLIES IS THAT IN ORDER TO GET THESE WHAT ARE CALLED ORGANIZING REGIONS FOR SOURCES OF SIGNAL THAT ARE IN THE FOREBRAIN TO ACTUALLY BE ESTABLISHED, YOU NEED THE MESENCHYMAL AS THE FIRST STEP. OWNER THE MESENCHYME IS ACTING DIRECTLY ON THE FOREBRAIN STEM CELLS WE'LL COME BACK TO. BUT WHAT'S KEY IS THAT IN ORDER TO ESTABLISH THESE SIGNALING CENTERS, ARE THE PRODUCED SIGNALS ARE THOUGHT TO ACT SECONDARILY ON THOSE CELLS. THE -- THIS IS FOR PATTERNING CLASSES OF RADIAL GLIAL -- FROM THE DORSAL FOREBRAIN WHICH MAKES THE CORTEX. SO WE'VE IDENTIFIED A GENE FROM AN ARRAY CALLED [INDISCERNIBLE] I WON'T TELL YOU WHAT THAT GENE IS BUT IT'S VERY SPECIFICALLY EXPRESSED IN THE MG VERSUS THE LG. IF WE HAVE -- ONLY, M IS EXPRESSED IF WE GIVE THE EPITHELIUM THE MESENCHYME -- IS EXPRESSED. IT'S TURN OUT THAT M -- IS SHOWN IN A BEAD EXPERIMENT HERE WHAT THIS SUGGESTS IS A TWO STEP MECHANISM OF THE MESENCHYME ELICITING FGF78 EXPRESSION ACTING ON M EXPRESSION THAT YOU SUBPATTERN THIS DISTINCT COMPARTMENT OF MG STEM CELLS WHICH ARE GOING TO MAC CORED CO-INTERNEURONS. WE'LL CESS THAT GENETICALLY, WHAT WE SEE IS MBIP EXPRESSION IS DIMINISHED IN WHAT REMAIN IN THE MEDIAL GANGLIONIC IN THE HYPER MORPHIC FG8 MUTANT. THIS DISTINCT -- SOME OF WHICH WILL MAKE INNER NEURONS AND STRIETLE NEURONS YOU USE THE MESENCHYME TO SET UP THE CENTER THAT THEN GO ON TO INFLUENCE RADIAL GLIAL IDENTITY AND CAPACITY TO GIVE RISE TO DIFFERENT NEURONS AND DIFFERENT FAITHS. NOW YOU SEE THAT THE MESENCHYME IS ACTUALLY ONLY OPPOSED TO ONE ASPECT OF THE STEM CELLS AND THAT IS THEIR BASAL DOMAIN WHERE THEY MAKE THESE GLIAL, THESE [INDISCERNIBLE] AND THE APICAL DOMAIN DOWN HERE AT THE VENTRICLE ZONE IS MOLEALLY DISTINCT. NOW WHAT I'M GOING TO SHOW YOU IS IF I SIMPLY MOVE THE MESENCHYME FROM THE BASAL DOMAIN TO THE APICAL DOMAIN, I CAN COMPLETELY DISRUPT RADIAL POLARITY. THIS IS NO LONGER EXPRESSED AT THE JUNCTIONS BETWEEN THE APICAL DOMAINS OF THESE CELLS. THE RADIAL ORIENTATION OF THESE PROCESSES THAT ARE HERE LABORED IS COMPLETELY DISREPRESENTED. AND FINALLY THE PLACEMENT OF CILIA RECOGNIZE WITH THIS ALPHA TUBULIN OR THE SIGNALING THAT DISTINGUISHES THE APICAL FROM THE BASAL DOMAIN IS COMPLETELY DISRUPTED. SO WE DON'T KNOW WHAT THE SIGNALS ARE THAT ARE COMING FROM THE MESENCHYME BUT WE DO KNOW THAT FOR ESTABLISHING AND MAINTAINING THE POLARITY OF THESE CREWS, THE MESENCHYMAL SIGNALS ARE KEY FROM AN EARLY AGE. WHETHER WE THOUGHT ABOUT THIS AND WE LOOKED AT THIS SIMPLE DIAGRAM WE REALIZED THERE WAS ANOTHER SOURCE OF SIGNALS THAT WE HAD FAILED TO CONSIDER. THAT IS THAT THESE ARE SECRETED SIGNALS OR CELL CONTACT SIGNALS COMING FROM THE MESENCHYME. THERE COULD BE SIGNALS COMING FROM THE -- WHAT WE THOUGHT WHAT WE KNEW ABOUT THE SIGNALING THAT WAS NECESSARY TO ESTABLISH AND MAINTAIN RADIAL GLIAL PROGENITORS AND TO DRIVE NEUROGENESIS FURTHER COULD NOT SIMPLY BE MANAGED DIC MULL SIGNALS. THERE'S A WAY OF MAKING THESE SIGNALS DYNAMIC. WE THOUGHT THAT MIGHT BE ACTUALLY SERVED BY THE AMNIOTIC FLUID VERY EARLY ON OR THE SPINAL FLUID. WHAT WE DID WAS WE ASKED WHETHER OR NOT AMNIOTIC FLUID AND CSF ARE DYNAMIC SOURCES OF SIGNALS DURING FOREBRAIN STEM CELL SPECIFICATION. WE HAD TO FIGURE OUT A WAY TO DO FROM VERY TINY EMBRYO IN UTERO. WHAT YOU SEE HERE MARKS ARE JUST BANDS THAT SHOW UP DIFFERENTLY IN 85 AMNIOTIC FLUID VERSUS E105 VERSUS E14CFS, ETCETERA. SO THERE SEEMS TO BE DYNAMISM BETWEEN AGES. WHAT WE ALSO DID WAS WE TOOK E105CFS JUST TO MAKE SURE THAT THIS JUST WASMENTED TELLING US ABOUT INDIVIDUAL VARIABILITY. WHAT YOU SEE HERE IN THESE EXAMPLES IS THERE'S A REMARKABLE CONSISTENCY BETWEEN INDIVIDUALS OF THE SAME AGE OF THE PROTEIN COMPONENTS IN THE CSS. NOW THE NEXT THING WE ASKED WAS WHETHER OR NOT THESE DIFFERENT AGES OF CSF OR AMNIOTIC FLUID COULD ACTUALLY MAINTAIN AGE SPECIFIC EXPRESSION OF GENES THAT DISTINGUISH RADIAL GLIAL PROGENITORS. WHAT WE SAW AGAIN AND ALL YOU NEED TO KNOW IS THIS IS E10CSF ACTING ON E105 EXPLANTED FOREBRAIN EPITHELIUM. WHAT YOU SEE IS THAT THE PRIMARY EXPRESSION OF ALL OF THESE GENES THAT ARE ACTUALLY ASSOCIATED WITH FOREBRAIN RADIAL GLIAL IS MAINTAINED OPTIMALLY BY E105CSF. 85AF AND 145AF CAN'T DO THIS. FOX G1 WHICH IS AN EARLY DETERMINANT ACTUALLY DOESN'T HAVE THE SPECIFICITY WHICH MAKES SENSE. SO WHAT THIS SAYS IS THERE'S SOMETHING SPECIAL ABOUT THE H MAX CSF THAT HAS THE SIGNALS THAT ARE NECESSARY TO MAINTAIN THE IDENTITY OF RADIAL GLIAL STEM CELLS. NOW THE NEXT THING IS WE WANTED TO KNOW WHETHER OR NOT THIS CHANGE FROM BEING THE FOREBRAIN EVEN DERM VERSUS THE TEN FIVE WHEN IT'S CLOSED. IT'S CHANGED FROM AMNIOTIC FLUID TO CSF BECAUSE CLEARLY THIS STUFF CAN'T MAINTAIN THE IDENTITY OF RADIAL GLIAL CELLS. WHETHER OR NOT THIS WAS ESSENTIAL FOR AN EARLIER STEP OF SPECIFYING FROM THESE PRIMITIVE NEURAL PROGENITOR THE RADIAL GLIAL. THE FIRST THING WE DID WAS BASICALLY SWAPPED AF AND CSF IN VITRO WITH EITHER ISOLATED E85 FOREBRAIN ECTO DERM E105. WE USED A SOX 2EFP REPORTER JUST FOR EASE. SOX 2 IS EXPRESSED IN ALL FOREBRAIN STEM CELLS FOR EARLY NEUROPROGENITORS AND EPIDERMAL STEM CELLS OR RADIAL GLIAL. AND WHAT WE SEE IS IF YOU USE E105CSF ON AN EH5 FOREBRAIN EPITHELIUM, YOU ACTUALLY ELIMINATE ALL OF THE NEUROPROGENITORS. THERE'S ACTUALLY TISSUE HERE AND THEY BECOME ECTO DERMAL. THEY EXPRESS ECTO DERMAL KERATIN. ACSF WHICH IS JUST THE ARTIFICIAL CSF AND DOESN'T HAVE THE PROTEINS IN IT, DOES A SIMILAR THING ONLY THE CELLS ACTUALLY DEGENERATE, THOUGH. IF WE DO THE CONVERSE EXPERIMENT, E105CSF ACTUALLY PROMOTES VOCAL EXPRESSION OF SOX 2 IN WHAT ARE FOCUSSAL ACCUMULATIONS OF RADIO GLIAL. WHAT THIS TELLS US THERE'S A DIFFERENTIAL CAPACITY FOR SIGNALS IN THE 85AF TO MAINTAIN EARLY ECTO DERMAL PROGENITORS VERSUS THE E105CSF TO MAINTAIN AND TO PROMOTE THE RADIAL GLIAL IDENTITY. SO WHAT ARE THE SIGNALS? THE FIRST THING WE LOOKED AT WAS RETONEIC AGENT WITH A CELL LINE HAT COULD DETECT CONCENTRATION OF RA IN PHYSIOLOGICAL LEVELS. THIS IS JUST SHOWING THIS IS ACTUALLY A BEAUTIFULLY LINEAR -- TEN TO THE MINUS LEVEL AND TEN TO THE MINUS 6. WHAT WE SEE IS THAT THE CONCENTRATION OF RA IS VERY LOW IN THE AMNIOTIC FLUID AND IT GOES UP DRAMATICALLY AS CORTICO NEUROGENESIS PROGRESSES. AND WHAT WE ALSO SEE IS IN THE OPEN TUBE THERE'S NO RA SIGNALING IN THE FOREBRAIN EVEN THOUGH IN THE CLOSE NODES DOWN IN THE SPINAL CORD THIS IS AN RA REPORTER LINE THAT WE USED IN THE DETAIL. THIS IS THAT SAME REPORTER TWO DAYS LATER. RA IS NOW BEING PRODUCED BY THE MESENCHYME AND WE SEE IT SIGNALS TO THESE RADIAL CELLS BUT NOT THE NEURONS. AND SIMILARLY YOU SEE THAT THE EXPRESSION OF THE RA SYNTHETIC ENZYME IS SEEN MOST SIGNIFICANTLY IN THE FRONT OR NASAL MESENCHYME. NOW FOUR DAYS LATER WE SEE FAR MANY MORE RADIAL GLIAL. THESE ARE RADIAL GLIAL THAT ARE RA ACTIVATED. WE SEE THE KEY SYNTHETIC ENZYMES FROM MAKING RETINOIC ACID ARE EXPRESS IN THE MENINGES. THIS IS THE SITE OF RA SYNTHESIS AND IT'S ACTUALLY RELEASED INTO THE CSF TO DIFFUSE AND ACTIVATE A MUCH MORE BROAD POPULATION OF CELLS. NOW THE OTHER THING WE'LL END WITH IS THE BMP'S. WHAT WE SAW TO OUR NOT SURPRISINGLY IS THAT BMP SIGNALING IN EARLY NEURAL ECTO DERM IS LIMITED. THIS IS IDENTIFIED WITH -- WE KNOW THAT BMP IS ACTUALLY KEY FOR AND IS SEEN PRIMARILY LIMITED TO THE RADIAL GLIAL. THIS IS THE QUANTIFICATION OF THE FREQUENCY OF PIECE IN THAT ACTIVATED CELL -- AS THESE AGES. WHAT WE DID FIRST OF ALL THINKING THAT POSSIBLY WHAT WAS GOING ON WAS THAT BMP CONCENTRATIONS COULD BE HIGH IN THE AMNIOTIC FLUID OR LOW IN THE AMNIOTIC FLUID AND HIGH IN THE CSF. WE ACTUALLY GAVE E10CFS KNOWINGEN WHICH IS ANTAGONIST. WHAT WE SAW IS WE COULD RESCUE THESE STEM CELL PHENOTYPE. WE THOUGHT WE'LL MEASURE THE BMP CONCENTRATION. SO WE DID A SIMILAR. WE DESIGNED A SIMILAR CELL LINE. IT DEFECTS IN A LINEAR FASHION THE BMP CONCENTRATIONS IN THE PHYSIOLOGICAL RANGE. SO THIS IS A GOOD TOOL. AND WE MEASURED THE MP CONCENTRATIONS AND WE GOT A SURPRISE. THE SURPRISE WAS THE BMP CONCENTRATIONS WERE ACTUALLY VERY HIGH AT E85 AND THEY DROPPED, AND THIS IS ACTUALLY A SIGNIFICANT DIFFERENCE WE'VE ACTUALLY ADDED DATA. THEY DROP DOWN TO LOWER LEVELS IN CSF AND ARE MAINTAINED AT LOW LEVELS. THIS IS SOMEWHAT OF A PARADOX. WHAT WE'RE THINKING ABOUT IS ACTUALLY THAT BMT SIGNALING AT THIS EARLY AGE IS ACTUALLY OPPOSED BY THE NEURAL CREST MESENCHYME THAT'S ARRIVING AND IT IS KNOWN TO EXPRESS NOGGIN AND OTHER BMP AGONIST AT HIGH LEVELS. BASICALLY WHAT YOU'RE DOING WITH THE MESENCHYME, AGAIN WE'RE ADDING THE MESENCHYME BECAME IN IS YOU'RE ANTAGONIZING THE HIGH LEVEL OF BMP AND MAINTAINING THE NEUROGENIC IDENTITY OF THE FOREBRAIN NEURAL EPITHELIUM. AT LAYER STAGES WHAT WE THINK IS GOING ON IS BMP IS ACTUALLY AVAILABLE AT LOWER LEVELS BUT NOGGIN EXPRESSION EITHER DECLINES OR NOGGIN BECOME LESS EFFECTIVE IN ANTAGONIZING BMP OR OTHER TGF-BETA SIGNALS AND THAT RESULTS IN THE ABILITY TO ACTIVATE -- BY PHOSPHORYLATION AND INFLUENCE RADIAL GLIAL IDENTITY AND DIFFERENTIATION. SO I THINK WHAT I'VE ENDED NOW IS IN THE STEM CELLS ARE THESE SOX 2 AND MEIS1 CELLS THE RADIAL GLIAL. KEY SOURCE OF SIGNAL IS THE MESENCHYME FOR BOTH STEM CELLS BUT YOU ADD THE AF AND THE CSF FOR AT LEAST THE FOREBRAIN. AND NOW WE'RE BEGINNING TO RECOGNIZE THAT ACTUALLY THE AMNIOTIC FLUID HAS A REALLY SIGNIFICANT INFLUENCE ON THE OLFACTORY EPITHELIAL STEM CELLS AS WELL. AND WHAT ARE THE SIGNALS? WE KNOW IN EACH CASE, RA, FDRA, FBMP ARE KEY SIGNALS FOR DEFINING BOTH OF THESE STEM CELL POPULATIONS AND THEIR PROGENY. AND SO WHAT I WOULD LIKE TO LEAVE YOU WITH IS THAT THE SPECIFICATION OF BOTH OF THESE STEM CELL CLASSES AT THE EARLY POINTS OF ACTUAL MAKING THE TRANSITION BETWEEN EITHER A NEUROGENIC ECTO DERM A NEURALLIZED ECTO DERM AND NEURAL EPITHELIUM TO GIVE RISE TO THE OE NEURONS OR FOREBRAIN NEURONS RELIES ON A SHARED MECHANISM BY WHICH MESENCHYME PROVIDES LOCAL SIGNALS THAT REGULATE POLARITY AND OTHER ASPECTS OF THE TRANSCRIPTIONAL CODE NECESSARY TO DEFINE CELL IDENTITY. AND THESE FLUIDS COMPARTMENTS ARE ACTUALLY DISBURSING SIGNALS MUCH MORE WIDELY BUT THEN HELP ACTUALLY ACTUALIZE THE PROLIFERATIVE AND DIFFERENTIATION CAPACITIES OF DISTINCT COMPARTMENTS OF CELLS. BY THEN I'LL END BY THANKING THE PEOPLE WHO DID THE WORK. -- DEFINE THE PHYSIOLOGICAL PROPERTIES. IN THE NEUROSCIENCE CENTER I HAD THE GREAT PLEASURE OF WORKING WITH -- WHO UNFORTUNATELY IN SEPTEMBER DIED VERY PREMATURELY AND SURPRISINGLY. AND SO IN TALKING ABOUT THIS WORK, MUCH OF IT WAS DONE AND IT'S VERY SAD TO RECOGNIZE HER LOVELY VOICE AND HER BRILLIANT INTELLECT IS NO LONGER AVAILABLE TO US. AT HUH VERT IN THE MOLECULAR CELL -- HARVARD IN THE MOLECULAR BIOLOGY DEPARTMENT [INDISCERNIBLE] PROVIDED MANY HELPFUL AND AT THE UNIVERSITY OF ARIZONA -- AT MY LABORATORY AT UMC. WITH THAT I'LL MAKE ANY QUESTIONS YOU MIGHT HAVE. THANK YOU. [APPLAUSE] >> [INDISCERNIBLE] >> IT MAINTAINS THROUGHOUT LIFE. WHAT'S BEEN PUBLISHED ON THIS IS ACTUALLY A STATE HELP. SO AFTER CORTICO RADIAL GLIAL ARE DISTINCT FROM THE DAY BEFORE. WHAT WE FOUND IS THAT IGF2 LEVELS DRAMATICALLY INCREASE AND THEN DROP. AND WHAT WE DID WAS BOTH GENETICALLY AND BY DOING THIS SORT OF SLOPPING EXPERIMENT, WE SHOWED THAT THE IDEA OF TWO CONCENTRATION CHANGES IN CSF ARE ABSOLUTELY CRITICAL FOR MAINTAINING THE RADIAL GLIAL CAPACITY OF THE FOREBRAIN, OF THE CORTICO RADIAL GLIAL TO ACTUALLY GENERATE CORTICO NEURONS. SO IF WE GENETICALLY TAKE THE IGF2 RECEPTOR, WE REST THE CELLS BETWEEN THE RADIAL GLIAL AND THE TRANSIT AMPLIFYING STAGE. IF YOU -- YOU COMPLETELY ABOLISHED NEUROGENESIS. IF WE GIVE IT THE RIGHT CFS WE'RE MAINTAINING KNEE OH GENESIS BEAUTIFULLY. IF WE ABSORB IGF2 FROM AGE SPECIFIC CSF YOU WOULD ABOLISH NEUROGENESIS. IF WE ADD IT BACK TO THE OTHER CFS. THERE SEEMS TO BE DYNAMISM THROUGHOUT LIFE. AND ONE OF THE THING THAT'S REALLY INTERESTING IS WE THINK THIS ACTUALLY MAY EXTEND TO DYNAMICALLY REGULATING CSF TO PUT THE BRAKES ON THE STEM CELLS THAT ARE RETAINED IN THE VENTRICULAR ZONE DURING LIFE. ONE WAY THAT WE TESTED THAT WAS WE GOT CSF FROM NEUROBLASTOMA PATIENT THAT HAD VERY RAPID PROGRESS OF THE TUMOR. THE IDF CONCENTRATIONS GO UP AND IN OUR ASSAYS YOU ACTUALLY PROMOTE NEUROGENESIS. SO IT DOES SEEM LIKE THROUGHOUT THE ENTIRE LIFE SPAN THAT THE REGULATION OF THE CSF COMPARTMENT IS ABSOLUTELY KEY. WHAT WE THINK THOUGH, THIS REFLECTS TWO THING. ONE IS THE MENINGES AND THAT IS RELATED TO THE NEUROCREST MESENCHYME INITIALLY. WE THINK THAT THE CONTACT SIGNALING AT THE BASAL DOMAIN IS ACTUALLY RETAINING IDENTITY OF THE PROGENITOR. >> THE SOURCE OF THE EARLY STAGE CSF IS THAT PROBABLY THE FACTORS THAT ARE SECRETED INTO IT ONCE THE NEUROTUBE CLOSES SO YOU HAVE A CLOSED SPACE ARE ACTUALLY COMING FROM THE NEUROEPITHELIUM ITSELF. AND ONE OF THE THING IS THAT WE KNOW THAT THERE ARE LOCAL SOURCES, THERE ARE PATCHES OF CELL THAT EXPRESSION BMP, SONIC HEDGE HOG AT GF8, ETCETERA. AND IT WAS THOUGHT THAT THAT WAS ACTING ONLY LOCALLY BUT THAT DOESN'T MAKE SENSE BECAUSE WE KNOW SIGNALING GETS DISTRIBUTED. BUT THEN LATER ON WHEN THE MENINGES ARE MADE AND THE CORE PLEXUS ARE MADE WE SEE THE PEPTIDE HORMONE LIKE IGF2 ARE IN EITHER THE MENINGES OR THE CORTICOID PLEXUSOR ENZYME LIKE RETINOIC ACID. WHAT HAPPENS IS YOU SHIFT THE SOURCE FROM THE PARENCHYMA ITSELF OF THE NEUROEPITHELIUM EARLY TO THESE ACCESSORY TISSUES LATER ON. SO THERE'S A DYNAMISM BOTH IN WHERE THEY COME FROM. >> [INDISCERNIBLE] >> IT'S AT NINE FIVE BECAUSE BASICALLY THE CTS DOESN'T FORM UNTIL YOU CLOSE THE NEURAL TUBE AND IT'S NO LONGER ACCESSIBLE TO THE AMNIOTIC FLUID. AT THE TIME THE TUBE CLOSES IF WE PULL THE VFS FROM THE NINE FIVE EMBRYOS AND WE LOOK AT THE PROTEIN CONSTITUENTS THEY'RE DIFFERENT THAN THE AMNIOTIC FLUID. SO THEY'RE DIFFERENT. >> [INDISCERNIBLE] >> THERE'S TWO POSSIBILITIES. ONE IS DIFFUSION BECAUSE THEY'RE DIFFUSING THROUGH A FLUID FILLED SPACE. THE OTHER THING IS THAT THERE ARE CILIA ON THE RADIAL GLIAL, AND THOSE CILIA ARE THOUGHT TO HAVE IN A THERE ARE KEY PLACES FOR SIGNALING EVENTS TO HAPPEN, INCLUDING SIGNALING THROUGH SONIC AND POTENTIALLY THROUGH FGF'S AND BMP'S. ALSO -- SO IT'S VERY POSSIBLE THAT THE ACTUAL SEWER THEMSELVES ARE DISTRIBUTING THEM FURTHER. >> ON THE ONE HAND THE MESENCHYME IS VERY WORRIED ABOUT WHERE IT IS IN SPACE. >> RIGHT. >> TALKING TO THE OE. ON THE OTHER HAND NOW YOU'RE DUMPING STUFF ON THE CSF AND GIVING UP SPACIAL CONTROL. >> RIGHT. >> APPARENTLY WHY. >> I THINK THE KEY FOR THAT IS THAT IT'S A TWO STEP PROCESS. I THINK A LOT OF THE MESENCHYMAL SIGNALING HAPPENS AT THE INITIAL STAGES BEFORE YOU ACTUALLY AND WHILE YOU'RE CLOSING THE NEURAL TUBE. THE NEURAL CREST MESENCHYME LIES AS THE, AT LEAST MORPHOLOGICALLY THE ANTERIOR IMROI BECOMES RECOGNIZABLE AS THE PRESUMPTIVE FOREBRAIN. THE CREST MESENCHYME ARRIVES AT THAT POINT. IT'S OPPOSED TO THE -- AND PROBABLY WHAT WE HYPOTHESIZE IS AT THAT POINT THE SIGNALING WHICH IS KEY FOR THE FOREBRAIN IS LOCAL. THAT THE AF SIGNALS ARE BASICALLY MAINTAINING NEUSH GENIC PRECURSOR IDENTITY WITH STEM CELL FATE. AS SOON AS THE, AS THE NEURAL TUBE CLOSES AND THE FOREBRAIN BEGINS TO EXPAND, YOU'VE SET UP ORGANIZATION BY VIRTUE OF WHERE THE MESENCHYME IS AND NOW YOU HAVE TO READ THAT UP BY DISTRIBUTING SIGNALS THAT MAINTAIN PROLIFIC CHARACTERIZATIONS OF SUBSELLS THAT ARE PATTERNED OR ENHANCE THAT WIDELY. THE CFS BECOMES AN EFFICIENT WAY OF DOING THAT. WHAT YOU DO TO GET DIFFERENCES IN THE PACE OF PROLIFERATION FROM DIFFERENT CLASSES OF RADIAL GLIAL THAT HAVE BEEN PATTERNED IN THE AP AND MEDIAL COLLATERAL DOMAINS IS YOU HAVE DIFFERENT SIGNALS AVAILABLE. AND THAT CAN ALSO DISTRIBUTE DIFFERENTIATION SIGNALS AS YOU'RE BEGINNING TO GENERATE POST MITOTIC NEURON. THIS IGF STORY THAT I CITED SEEMS TO BE AN EXAMPLE OF THAT. SO WHAT WE'RE DOING NOW IS WE'RE ACTUALLY BY MASS SPECK WE'RE GOING THROUGH AND IDENTIFYING ALL OF THE SIGNALS THAT ARE AVAILABLE AT THE VARIOUS TIMES AND BEGINNING TO GET CANDIDATES THAT WOULD ACTUALLY DO THIS. AND THEM TRYING TO PUT THAT TOGETHER WITH. >> AND THE LOWER LEVEL WHERE WE HAVE THE ACTUAL STEM CELLS ARE IN THE LATERAL PART. YOU WILL SAY THAT FGS HAS AN IMPORTANT ROLE IN KEEPING HIGH COY SO FGC IS EXPRESSED ALONG THE AREA IN THE LATERAL AREA AND NOT THE MEDIAL AREA SO THE HIGH CONCENTRATION -- >> SO FGS8 AS THE FRONTAL NASAL PROCESS CLOSES, IT IS EXPRESSED LATERALLY BUT ALSO IT EXTENDS MEDIALLY UP INTO THE EPITHELIUM. SO BASICALLY IT'S COMING SORT OF FROM -- >> THE HIGHEST CONCENTRATION OF FGS8 ARE ACTUALLY -- >> WHEN WE DID THE INSIDE TEASE AND WE DID THIS IN 2000 WE PUBLISHED THIS WE HAD GFS MORE INTENSE THREE MEDIALLY. THAT'S SOMETHING WE HAVEN'T BEEN ABLE TO FIGURE OUT. BUT IT DOES SEEM THAT GF8 IS FROM THE GENETICS IT TELLS US THAT INDEED FGF8 CONCENTRATION IS CONTROLLING THE LEVELS OF SOX 2 BECAUSE THE FGF8 HYPER MORPH DOESN'T DISRUPT -- IT DISRUPTS THE LEVEL OF SOX 2 AND THE EXPRESSION OF ASCL1. >> I SHOW YOU -- >> I THINK THE OTHER THING IS THAT REMEMBER THAT WHERE THE SIGNAL IS IN THE EVEN TEAM, NOT IN THE RHESUS KIND BUT THE EPITHELIUM, IT DOESN'T SAY THE EPITHELIUM WILL ACT THERE. ONE OF THE THING IT'S PROBABLY DIFFUSING LOCAL 8 EVEN INTO THE CAVITY. THE MESENCHYME IT'S DIFFERENT BECAUSE THERE IS A CONTACT MEDIA OF THE MESENCHYMAL ACTION WHICH INCLUDES FGS78 THE LOCALIZATION BECOMES LESS IMPORTANT SO THAT'S A POTENTIAL ANSWER TO THIS. OKAY. >> [INDISCERNIBLE] >> WE'D LIKE TO LOOK AT THAT. THE QUESTION IS WHETHER OR NOT WHEN YOU DELAY A NEUROTUBE CLOSURE YOU CAN DELAY IT RATHER THAN COMPLETELY DISRUPT IT. [INDISCERNIBLE] WHETHER OR NOT THAT ACTUALLY DISRUPTS SIGNALING IN A SIGNIFICANT WAY AND CHANGES PRECURSOR SPECIFICATION. I THINK THE PROVISION ANSWER IS ONE WOULD IMAGINE THAT IT WOULD. BECAUSE EVEN IF THE SOURCES OF SYNAPSE THAT ARE ESTABLISHED IN THE FOREBRAIN ECTO DERM ARE CAPABLE OF RELEASING THE APPROPRIATE SIGNALS, THEIR CONCENTRATION IS GOING TO BE DIMINISHED. AND WHAT THIS ARGUES WHICH IS SOMETHING THAT I THINK EVERYBODY WAS AWARE OF BUT NOBODY COULD FIGURE OUT WAS THE EFFECTIVENESS OF THE SIGNALS LIKE FGF'S AND VMP'S AND SONIC AND RA WHICH ARE DIFFERENT ONES THAT HAVE MAINLY BEEN LOOKED AT THEIR EFFECTIVENESS IS MUCH FURTHER AFIELD FROM WHERE THEIR SOURCES ARE. SOME COULDN'T FIGURE OUT WHY THAT IS. WHAT WE WOULD ARGUE IS THAT SECRETING THEM INTO THE CSF IS THE KEY FOR DISTRIBUTING THEM TO ALL THE TARGET CELLS. SO IF YOU JUST LOWER THEIR CONCENTRATION SUFFICIENTLY OR RAISE THE OTHER CONCENTRATIONS [INDISCERNIBLE] YOU COULD DISRESULT THE SIGNALING. INDEED I THINK THE ONE THING IS THAT WE ACTUALLY WE TALKED ABOUT THIS BUT ONE PROBLEM IS A LOT OF NEUROTUBE CLOSURE MUTANTS THAT HAVE BEEN LOOKED AT ARE ACTUALLY THEIR MOSTLY CYTOSKELETAL MUTANTS. SO THE ABILITY TO ACTUALLY SORT OUT PROLIFERATIVE CHANGES OR DIFFERENTIATION CHANGES BECAUSE OF THE CSF CHANGES VERSUS THE MUTATION ITSELF WOULD BE DIFFICULT. TO ACTUALLY GET A MUTATION WE'VE LOOKED, TO GET A MUTATION WHERE YOU DISRUPT THE NEURAL TUBE CLOSURE BUT NOT DISRUPTING POLARITY AND CYTOSKELETAL MECHANISMS THAT REGULATE DIVISIONS AND DIFFERENTIATIONS IS VERY DIFFICULT. >> THANK YOU