GOOD AFTERNOON ITS MY PLEASURE TO INTRODUCE DR MARISA BARTOLOMEI OVER THE YEARS SHE MADE IMPORTANT CONTRIBUTIONS ENDOCRINE DISRUPTING AGENTS SUCH AS BPA, HER LAB HAS SHOWN THIS EXPOSURE IS ASSOCIATED WITH INCREASED LOSS OF IMPRINTING AND MAY AFFECT METABOLIC HEALTH ACROSS MULTIPLE GENERATIONS IN THE MOUSE THROUGH STABLE INHERITANCE OF DNA METHYLATION AT THE IMPRINTED LOCUS. Igf2. AND THIS REPRESENTS ANOTHER EXAMPLE OF TRANSGENERATIONAL EPIGENETIC INHERITTANTS THAT RESULTS IN DEVELOPMENTAL DEFECT. IN ADDITION TO STELLAR SCIENCE, MARISA HAS BEEN A GREAT SCIENTIFIC CITIZEN SERVING ON NUMEROUS EDITORIAL BOARDS AND IN ADDITION TO SERVING ON MANY NIH REVIEW PANELS OVER THE YEARS SHE'S PRESENTLY A MEMBER OF THE NIH COUNCIL OF COUNCILS COMMON FUND EVALUATION WORKING GROUP AND ON THE ADVISORY GROUP FOR EPIGENOMIC AND 4D NUCLEOME. THE TITLE IS WHY PARENTS MATTER, AND JOIN ME IN WELCOMING MARISA TODAY. [APPLAUSE] >> OKAY. FIRST, I WOULD LIKE TO THANK YOU ALL FOR HEADING OUT IN THE COLD WEATHER TO COME HERE, AND I WOULD ESPECIALLY LIKE TO THANK STEWART AND THE NIH FOR INVITING ME TO GIVE THIS LECTURE. I'VE HAD A GREAT DAY, SEEING A LOT OF FRIENDS AND HOPING YOU'LL BE ABLE TO GET A LOT OUT OF THIS LECTURE AND BE MORE APPRECIATIVE OF WHY YOUR PARENTS REALLY DO MATTER, AT LEAST YOU CAN GO HOME AND TELL YOUR CHILDREN. SO LET ME JUST START, WHAT I WOULD LIKE TO DO TODAY IS JUST GIVE YOU A LITTLE BIT OF BACKGROUND ABOUT IMPRINTING AND THEN TELL YOU A LITTLE BIT ABOUT OUR MOUSE MODELS THAT WE DEVELOP IN THE STUDY, HUMAN IMPRINTING DISORDERS AND TELL YOU TWO STORIES, ONE ABOUT SINGLE CELL IMAGING THAT WE DEVELOPED THAT WE'RE INCREDIBLY EXCITED ABOUT IN COLLABORATION WITH SOME GREAT NEW COLLEAGUES AT PENN, AND THEN ALSO I'LL TELL YOU A LITTLE BIT ABOUT SOME OF OUR STUDIES ON REPRODUCTIVE TECHNOLOGIES. THIS FIRST SLIDE HERE JUST GIVES YOU THE DEFINITION OF GENOMIC IMPRINTING WHICH IS THE UNEQUAL EXPRESSION OF THE TWO PARENTAL ALLELES OF A GENE. FOR MAMMALS WHO HAVE TWO SETS OF CHROMOSOMES, ONE THEY INHERIT FROM THE MOTHER, ONE THEY INHERIT FROM THEIR FATHER, MOST OF THESE GENES ARE EXPRESSED FROM BOTH ALLELES, BARRING SOME MUTATION. FOR IMPRINTED GENES THEY ARE EXPRESSED FROM THE PATERNAL ALLELE OR MATERNAL WITH OPPOSITE BEING SILENT, THEY ARE IMPRINTED. WE KNOW THERE ARE OVER 100 IMPRINTED GENES DESCRIBED IN MOUSE, AND MANY OF WHICH ARE CONSERVED IN HUMAN, AND SO WHAT WE'VE BEEN ABLE TO TO DO BECAUSE OF THE CONSERVATION BETWEEN MOUSE AND HUMAN WE'VE BEEN ABLE TO GO BACK AND FORTH IN STUDYING THEM, BETWEEN MOUSE AND HUMAN. SO WHAT HAPPENS, THE CONSEQUENCE OF IMPRINTED GENES IS THE MAIN CONSEQUENCE IS IN MAMMALS YOU REQUIRE BOTH A CONTRIBUTION FROM YOUR MOTHER AND A CONTRIBUTION FROM YOUR FATHER IN ORDER TO BE VIABLE. OTHER ORGANISMS YOU HAVE CARCINOGENESIS BUT IN MAMMALS YOU REQUIRE BOTH MATERNAL AND PATERNAL CONTRIBUTIONS. SO WHAT ARE THESE GENES THAT ARE IMPRINTED? WHEN WE FIRST IDENTIFIED IMPRINTED GENES OVER 20 YEARS AGO, WE SAW THESE GENES WERE MOSTLY INVOLVED IN GROWTH, THAT THEY WERE IMPORTANT FOR FETAL GROWTH. BUT NOW WE KNOW THAT THESE GENES HAVE LOTS OF ROLES IN DEVELOPMENT SO IN ADDITION TO EITHER ENHANCING OR SUPPRESSING GROWTH THEY ARE INVOLVED IN MEMORY, IN COGNITION, IN BEHAVIOR, POSTNATAL ENERGY HOMEOSTASIS AND A VARIETY OF PROCESSES. SO THESE GENES DO HAVE VERY CENTRAL ROLES IN DEVELOPMENT. WHAT WE ALSO KNOW IS THESE GENES THEN ARE MONO ALLELICALLY EXPRESSED, AND WE KNOW TOO MUCH IS BAD SO IF YOU HAVE A LOSS OF IMPRINTING SCENARIO WHERE YOU HAVE BOTH ALLELES BEING EXPRESSED, THAT'S BAD. AND WE KNOW IF YOU JUST HAVE A SINGLE MUTATION OR SOME TYPE OF OTHER EFFECT WHERE YOU DON'T HAVE ANY EXPRESSION AT ALL, THAT'S BAD. SO HERE ARE SOME EXAMPLES ON THIS SLIDE. I HAVE SOME MOUSE MUTATION EXAMPLES OF IMPORTANCE OF IMPRINTED GENES AS WELL AS HUMAN SYNDROMES. SO ON THE LEFT IS AN EXAMPLE OF AN IMPRINTED GENE, REALLY INTERESTING IMPRINTED GENE CALLED GRAB 10, INVOLVED IN GROWTH, IT'S A REALLY UNUSUAL IMPRINTED GENE IN THAT IT STARTS OUT BEING EXPRESSED FROM THE MATERNAL ALLELE IN SOMATIC TISSUES BUT THEN LATER ON IN DEVELOPMENT IN NEURONAL LINEAGE IT FLIPS TO THE PATERNAL ALLELE, A REALLY UNUSUAL IMPRINTED GENE. WHEN THIS GENE IS KNOCKED OUT, OR DELETED, IN MICE, AND IS TRANSMITTED BY THE MOTHER, SO THAT THE MOTHER'S ALLELE IS DELETED, BUT THE FATHER'S ALLELE IS SILENT YOU SEE OVERGROWTH OF THE MOUSE FETUS WELL ACTIVE TO WILDTYPE LITTER MATE. HERE IS CDCN1C A GROWTH REGULATORY GENE INVOLVED IN A NUMBER OF DISORDERS INCLUDING BECKWITH-WIEDEMANN SYNDROME WHICH I'LL DESCRIBE IN A SECOND BUT IN THIS CASE DELETION OF CDCN1C CAUSES PLACENTAL PATHOLOGY, SO ABERRANT MORPHOLOGY UPON DELETION. THAT'S MOUSE. WHAT ABOUT HUMANS? THERE ARE A NUMBER OF IMPRINTING SYNDROMES DESCRIBED THROUGH THE YEARS, A FEW OF WHICH ARE SHOWN UP HERE, SO, BECKWITH-WIEDEMANN SYNDROME, THESE ARE RARE. HERE IS AN EXAMPLE WHICH IS AN OVERGROWTH SYNDROME, AND THERE ARE TWO NEURAL BEHAVIOR SYNDROMES, PRADER-WILLI AND ANGELMAN, WHICH ARE SEVERE AND INVOLVE IMPRINTED GENES EITHER BEING DELETED OR IMPRINTED EXPRESSION BEING ABERRANTLY CONTROLLED. SO WHAT DO WE KNOW ABOUT THE REGULATION OF IMPRINTED GENES? THAT'S WHAT MY LAB AND NUMEROUS OTHER LABS INCLUDING CARL FIFER HERE AT NIH ARE INTERESTED IN REALLY THE MECHANISM OF THIS IMPRINTING CONTROL. SO WHAT WE KNOW IS THAT THESE GENES ARE ACTUALLY FOUND IN CLUSTERS OF THE GENOME SO THERE MAY ONLY BE FEWER THAN 200 IN MOUSE BUT MOST OF THESE IMPRINTED GENES ARE CLUSTERED, IN ONE TO TWO MEGABASE CLUSTERS WHERE YOU HAVE MA TERNALLY EXPRESSED GENES, PA TERNALLY EXPRESSED GENES AND WE KNOW FOR THESE CLUSTERS THEY ARE CONTROLLED BY A DISCRETE ELEMENT, DISCRETE ELEMENT THAT MAY JUST BE A COUPLE HUNDRED BASES, UP TO A COUPLE KB, THAT ARE DIFFERENTIALLY DNA METHYLATED. AND SO DNA METHYLATION WHICH IS IN THIS CASE I'VE SHOWN IS THE ADDITION OF A METHYL GROUP TO THE CYTOCEINE, THIS DIFFERENTIAL METHYLATION FOR THESE CLUSTERS IS CRITICAL FOR THE PROPER REGULATION, SO IF THIS ELEMENT IS DELETED YOU SEE LOSS OF IMPRINTING AND LOSS OF PROPER EXPRESSION, IF THERE'S SOME OTHER PERTURBATION IN THE SYSTEM THAT LEADS TO CHANGES IN THE DNA METHYLATION STATE OF THIS DISCRETE ELEMENT YOU SEE ABERRANT EXPRESSION OF THESE IMPRINTED GENES, SO THIS ELEMENT IS VERY KEY TO THIS, YOU KNOW, POSSIBLY 2 KB ELEMENT IS KEY TO THE EXPRESSION OF A SET OF GENES, POSSIBLY OVER TWO MEGABASES IN SIZE. SO JUST A LITTLE BIT MORE BACKGROUND, THERE ARE A COUPLE DIFFERENT WAYS THAT HAVE BEEN DESCRIBED THAT THESE IMPRINTED GENE CLUSTERS ARE REGULATED, AND I JUST WANT TO PRESENT ONE WAY THAT'S ACTUALLY USED MORE FREQUENTLY BEFORE I GO MORE SPECIFICALLY INTO H19 AND Igf2. THIS IS THE LONG NON-CODING RNA MECHANISM OF IMPRINTED GENE REGULATION, IT REALLY WAS ONE OF THE FIRST EXAMPLES OF HOW LONG NON-CODING RNAs COULD THEMSELVES BE REALLY CRITICAL FOR GENOME REGULATION. THIS PARTICULAR CLUSTER WAS ORIGINALLY IDENTIFIED BY DENISE BARLOW, AND Igf2R WHICH IS EXPRESSED FROM THE MATERNAL ALLELE WAS ONE OF THE FIRST THREE GENES THAT WERE DESCRIBED AS IMPRINTED. SO HOW DOES REGULATION AT THIS LOCUS WORK? YOU HAVE Igf2 EXPRESSED FROM THE MATERNAL ALLELE AS WELL AS THESE SOLUTRANSPORTER GENES AND ON THE PATERNAL ALLELE IS A LONG NON-CODING RNA THAT'S CALLED THE AIR N NON-CODING ANN, OVER 100 KILOBASES IN END LA. THE KEY TO IMPRINTATION OF LOCUS IS IMPRESSION OF THIS GENE WHICH IS REGULATED BY THIS IMPRINTING CONTROL REGION SO THE PROMOTER OF AIR N RESIDES WITHIN THIS COUPLE KB IMPRINTING CONTROL ELEMENT, AND WHEN IT'S UNMETHYLATED, AIR N IS EXPRESSED, AND IT REPRESSES Igf2R AND THESE OTHER GENES IN SYS, POSSIBLY THROUGH RECRUITMENT OF EPIGENETIC MODIFIERS, REPRESSIVE PROTEINS THAT REPRESS THESE GENES. ON THE MATERNAL ALLELE THIS PARTICULAR ELEMENT IS METHYLATED, BECOMES METHYLATED DURING OOCYTE MATURATION, THAT METHYLATION IS SUFFICIENT TO SURGEON -- TURN OFF EXPRESSION OF THESE GENES, ENABLING THESE GENES TO BE EXPRESSED ON THAT LOCUS. SO IT'S ALL ABOUT THIS METHYLATION AND EXPRESSION OF THIS GENE. SO OF THE DOZEN OR SO IMPRINTED CLUSTERS THAT HAVE BEEN WELL STUDIED, IN THE MOUSE AND HUMAN, THIS IS THE MOST COMMON MECHANISM FOR REGULATING IMPRINTED GENE CLUSTERS. ONE USED FREQUENTLY EVOLUTIONARY THE MOST ANCIENT BECAUSE REMNANTS CAN BE FOUND IN PLACENTAL MA'AM MILLIONS IS THE H19 AND IFG 2 LOCUS, THE GROWTH FACTOR 2 GENE AND DELETION OF Igf2 GENE RESULTS IN UNDERGROWTH ARE NO VIABILITY, MICE ARE VIABLE BUT SMALL. H19 IN CONTRAST IS ACTUALLY A NON-CODING RNA, IT MAKES ABOUT A 2.2, 2.3 KILOBASE LONGER NON-CODING RNA, OUT OF THE FIRST AXON ENCODES A microRNA, STILL A QUESTION ABOUT WHETHER THE LONG NON-CODING RNA OR THE microRNA HAS A FUNCTION, BUT I THINK THAT WHEN THIS WAS FIRST STUDIED IN SHIRLEY'S LAB MANY YEARS AGO THE GENE WAS DELETED, NO OBVIOUS PHENOTYPE SO WE WERE SAD THAT MAYBE THE GENE DOESN'T HAVE A FUNCTION BUT I THINK IT'S VERY CLEAR NOW FROM WORK THAT'S BEING DONE IN THE SERIES OF DIFFERENT LABS THAT THIS NON-CODING RNA DOES HAVE IMPORTANT FUNCTIONS IN DEVELOPMENT AND BEYOND BUT THEY ARE VERY SUBTLE AND DIFFICULT DETECT UNLESS YOU'RE VERY CAREFUL IN A MOUSE MODEL. HOW DOES IMPRINTING AT THIS LOCUS WORK? WELL, THE IMPRINTING CONTROL REGION WHICH IS DIFFERENTIALLY METHYLATED ALSO SERVES AS AN INSULATOR. AND SO IT'S A CTCF DEPENDENT INSULATOR, SO H 19 IS EXPRESSED FROM THE MATERNAL ALLELE, Igf2 FROM THE PATERNAL, THEY SHARE ENHANCERS, AND WHAT'S KEY TO THE REGULATION IS THIS CTCF DEPENDENT INSULATOR, SO WHEN IT'S UNMETHYLATED THERE ARE FOUR CTCF BINDING SITES IN THE MOUSE, CTCF FINDS, PREVENTS Igf2 FROM ACCESSING THESE SHARED ENHANCERS, THAT DRIVES THE EXPRESSION OF H19. IN CONTRAST, ON THE PATERNAL ALLELE, THIS ELEMENT IS METHYLATED IN THE MALE GERMLINE, DURING GAMETO GENESIS, SUFFICIENT TO SHUT OFF H19, FREEING UP THE ENHANCERS TO DRIVE THE EXPRESSION OF Igf2, SO THIS HAS BEEN -- THIS MODEL HAS BEEN DEVELOPED THROUGH THE YEARS BY THE CONTRIBUTIONS OF THE TOMEN LAB, CARL FIFER'S LAB, MY LAB AND MANY OTHERS, IT'S BEEN A LOT OF WORK THAT LED TO THIS. WHAT WE CERTAINLY KNOW IS THAT IT ACTUALLY DOESN'T TAKE VERY MUCH TO DISTORT THE BALANCE OF EXPRESSION, CHANGE THE METHYLATION STATE AND RESULTING IN ABERRANT EXPRESSION OF THESE IMPRINTED GENES. SO WHAT I JUST WANTED TO DESCRIBE, WE'VE NOW ACTUALLY SPENT A LOT OF THE LAST 20 YEARS IN MY LAB BUILDING MOUSE MODELS TO ACTUALLY STUDY THIS IMPRINTED GENE REGULATION, AND NEVER REALLY ENTIRELY INTENTIONALLY, WE ACTUALLY HAPPENED UPON MOUSE MODELS THAT MIMICKED HUMAN IMPRINTING SYNDROMES, ONE OF THESE BEING SILVER-RUSSELL, MORE SO, BECKWITH-WIEDEMANN BUT I'M NOT GOING TO TALK ABOUT WORK WITH BECKWITH, I WANT TO CONFINE TO THIS MODEL AND USE THIS TO SHOW EXCITING SINGLE-CELL IMAGING WE'VE BEEN DOING IN OUR LAB, ALLELE-SPECIFIC IMAGING. SO THIS MOUSE MODEL AS I'LL DESCRIBE TO YOU IN A SECOND IS A GOOD MODEL FOR SILVER-RUSSELL. WHAT WAS THE GOAL IN THE ORIGINAL EXPERIMENT? ORIGINALLY WE WERE TRYING TO UNDERSTAND WHAT IT TOOK TO REPRESS H19 ON THE PATERNAL ALLELE, AND SO WE KNEW THAT THERE WERE A SERIES OF CpG DINUCLEOTIDES, SITES OF METHYLATION, ASSOCIATED WITH REPRESSION SO WHAT WE DECIDED TO DO WAS START TO MUTATE SOME OF THESE CpG DINUCLEOTIDES TO SEE WHAT WAS SORT OF THE CRITICAL POINT THAT WAS REQUIRED FOR REPRESSING THAT PATERNAL ALLELE SO WE HAD 60 CpGs TO WORK WITH, WHICH SHOULD WE START WITH, WE DECIDED TO START WITH THE ONES MOST CONSERVED ACROSS DIFFERENT STRAINS OF MICE AND SPECIES, IT TURNED OUT TO BE CpG BINDING SITES. WE MATE THE NINE CpG MUTATIONS, THEY ALLOWED CTCF TO BIND TO THE SEQUENCES, THEY WERE NOT IMPORTANT CONTACT SITES FOR CTCF BUT IF METHYLATED THEY BLOCKED CTCF FROM INTERACTING IN THE SEQUENCE. WHEN WE MUTATED AND TRANSMITTED PATERNALLY WHAT WE SAW CGCF BOUND, H19 WAS EXPRESSED, Igf2 WAS REPRESSED, WE ENDED UP WITH SMALL MICE, THEY LOOKED SIMILAR TO MICE THAT WERE DELETED FOR Igf2. AS I MENTIONED TO YOU, IT TURNED OUT THESE MICE WERE A PRETTY GOOD MODEL FOR SILVER-RUSSELL SYNDROME BECAUSE AFTER ABOUT A YEAR AFTER WE PUBLISHED THAT ORIGINAL STORY, THESE PAPERS CAME OUT, SILVER-RUSSELL SYNDROME OR RUSSELL-SILVER SYNDROME, IT DEPENDS WHAT SIDE OF THE OCEAN HOW YOU CALL IT, IS A SMALL FOR GESTATIONAL AGE, THE CHILDREN ARE BORN GREATER THAN THREE STANDARD DEVIATIONS BELOW MEAN BIRTH WEIGHT FOR THEIR AGE, AND THEY HAVE FAILURE TO THRIVE AND ASYMMETRIES AND OTHER TYPES OF CHARACTERISTICS. AND IT TURNED OUT THAT THE GROUP THAT HAD BEEN STUDYING THIS ABOUT BEEN STUDYING GENETIC LINKAGE BUT STARTED TO THINK ABOUT THE SMALL MICE AND IMPRINTING AND DECIDED TO LOOK AT H19 AND Igf2 IN THEIR PATIENTS AND THEY ACTUALLY FOUND THAT MANY OF THE PATIENTS HAD LOSS OF METHYLATION, DNA MET METHYLATION, LEADING TO BIOLOGIC H19, LOSS OF Igf2. SO THAT WAS VERY HELPFUL IN TRYING TO TRACK THAT. SINCE THEN IT'S SHOWN A VAST PROPORTION OF PATIENTS WITH IS I HAVE THIS METHYLATION LESION. OKAY. SO BACK TO THE MOUSE MODEL, WHEN WE GENERATED THIS MODEL, WE KNEW THAT WE HAD BIALLELIC EXPRESSION OF H19 BUT WE ALSO HAD A SMALL AMOUNT OF Igf2 PRESENT. SO RESIDUAL Igf2 PRESENT. AND SO IT WAS REALLY UNCLEAR TO US WHERE THE Igf2 WAS COMING FROM AND WHY THERE WASN'T COMPLETELY 50-50 H19 EXPRESSION, EQUAL FROM THE MATERNAL AND PATERNAL ALLELE. WE STARTED TO THINK ABOUT WHAT DOES THIS REALLY MEAN, WHAT IS BIALLELIC EXPRESSION LOOKING LIKE AT THE SINGLE CELL LOCUS? A WILDTYPE EXPRESSION, ONLY THE MATERNAL OF H19, MATERNAL ALLELE OF H19 IS EXPRESSED, AND THE MUTANT WE SEE BIALLELIC EXPRESSION, BUT IS IT BECAUSE THE MATERNAL AND PATERNAL ALLELE IS EXPRESSED IN EVERY CELL? OR IS IT BECAUSE YOU HAVE A MIXED POPULATION OF WILDTYPE LOOKING CELLS AND MUTANT CELLS, OR SOME COMBINATION OF THAT? AND WHY WE CARE ABOUT THAT IS BECAUSE IT HAS REALLY IMPORTANT IMPLICATIONS FOR THE MECHANISM OF WHAT'S GOING ON, AND IT ALSO IS THAT WE'RE LOOKING IN COMPLEX POPULATIONS OF CELLS, AND THERE MAY BE THAT CERTAIN CELL TYPES IN THE LIVER OR MUSCLE ARE EXPRESSING THE GENE WILDTYPE AND OTHER TYPES ARE EXPRESSING IT IN A MUTANT MANNER, AND THAT MAY TELL US SOMETHING ABOUT TISSUE-SPECIFIC CONTROL OF IMPRINTING AND WHAT THE PHENOTYPES IN OUR MICE OR THE HUMAN PATIENTS LOOK LIKE. SO WHAT WE REALLY LIKE TO DO IS LOOK AT LARGE POPULATIONS OF CELLS AT ONE TIME, BUT SINGLE CELLS. AND SO WE TRIED FOR A NUMBER OF YEARS TO GET ALLELE-SPECIFIC IN SITU HYBRIDIZATION TO LOOK AT H19 MATERNAL AND PATERNAL ALLELES, BUT THAT DIDN'T WORK FOR US, AND SO ALONG COMES SOME REALLY WONDERFUL COLLEAGUES THAT JOIN THE ENGINEERING DEPARTMENT AT PENN, AND EVERY PLACE NEEDS AN ENGINEERING DEPARTMENT BECAUSE THEY HAVE GREAT TECHNOLOGY AND THEY ARE AWESOME COLLABORATORS AND THEY WANT TO BRING THEIR TECHNOLOGY TO YOUR BIOLOGICAL QUESTIONS. AND SO ARJUN RAJ, ASSISTANT PROFESSORS THAT TWO WONDERFUL STUDENTS, WE UP FOR THE CHALLENGE OF IMAGING ON SINGLE-CELL BASIS, SO THE IDEA IS IF YOU HAVE TWO GENETICALLY DISTINCT, DNA POLYMORPHISM, RNA POLYMORPHISM, TWO GENETICALLY DISTINCT STRAINS OF MICE THAT HAVE POLYMORPHISMS, IN A CELL, CAN YOU DETERMINE WHETHER THE RNA THAT COMES, THAT YOU DETECT, COMES FROM EITHER THE MATERNAL ALLELE OR PATERNAL ALLELE, JUST BASED ON THESE POLYMORPHISMS? SO THE POLYMORPHISMS ARE OVER A LARGE REGION, BY USING SHORT OLEIGOTES THERE'S NOT ENOUGH SPECIFICITY. MARSHALL SAID LET'S TRY A DIFFERENT APPROACH, A TOE HOLD APPROACH. HERE IS THE POLYMORPHISM, SINGLE STRANDED, LET'S TAKE THE REST OF THE OLIGO AND TAKE ANOTHER PIECE OF DNA AND CREATE A DOUBLE-STRANDED, SO YOU HAVE A SINGLE STRAND WITH THE POLYMORPHISM AND DOUBLE-STRANDED WITH NO POLYMORPHISM. SINGLE-STRANDED PART WILL COME AND BIND TO YOUR RNA WHERE YOU HAVE THAT FAINT POLYMORPHISM, OKAY? IF IT WERE JUST THAT PIECE IT WOULD SLOT BACK ON AND OFF BUT THE FACT THAT YOU HAVE THIS DOUBLE-STRANDED PIECE HERE, ONCE THAT PIECE OF THE OLIGO BINDS TO RNA, KINETICS OF BINDING IS SUCH THAT THIS IS GOING TO MELT OFF AND THIS PROBE WILL THEN GO AND BIND TO THE REST OF YOUR RNA, AND IT HAS A FLOOR SO IT HAS A FLOOR THAT'S DISTINCT. WE HAVE FLOORS FOR THE BLACK SIX STRAIN, MATERNAL ALLELE, AND THEN WE HAVE A DIFFERENT PROBE WITH THE DIFFERENT FLOOR FOR THE OTHER ALLELE, POLY MORPHIC ONE, SAME THING GOES HERE. WHAT DOES THE EXPERIMENT LOOK LIKE? HERE IS A FIBROBLAST, AND WE FIRST TAKE GUIDE PROBES AND SO THIS HAS ONE FLOOR, IT WILL BIND TO EVERY H19 RNA IN THE TUBE, IN THE CELL, AND THEN WE ADD OUR MATERNAL SNP PROBE AND PATERNAL SNP AND LOOK FOR WHERE THE GUIDE PROBE CROSS HYBRIDIZES WITH EITHER THE PATERNAL PROBE OR THE MATERNAL PROBE BUT NEVER BOTH. OKAY? AND SO ONLY IN CASES WHERE THERE'S HYBRIDIZATION OF ONE OR THE OTHER AND THE GUIDE TO DO WE COUNT IT. IMAGING BY FLUORESCENCE BUT THE COMPUTER MAKES ALL THE CALLS. HERE IS A FIBROBLAST AGAIN. HERE IS THE HYBRIDIZATION. HERE IS THE PART OF THE CELL. HERE IS THE GUIDE PROBE CHANNEL. HERE IS THE ONE PROBE AND THE BLACK SIX PROBE CHANNEL, THE COMPUTER MAKES CALLS FOR THE GUIDE PROBE, THE CASS PROBE, BLACK SIX PROBE AND OVERLAPS AND SAYS THERE'S THESE MANY THAT ARE CAST, C7, THESE ARE BLACK SIX AND IT COUNTS AND GOES THROUGH THE CELLS AND IT DOES IT AUTOMATED BY GREAT PROGRAMMING SOFTWARE. HERE IS OUR TEST CASE. WE DO THIS CROSS, F1 HYBRID, CASTANEUS, BLACK SIX. YOU'LL SEE A FEW FIGURES, EACH LINE IS A CELL THAT'S BEEN QUANTIFIED. AND YOU CAN SEE THAT THE ORANGE CORRESPOND TO THE C7 CASTANEUS, THE BLUE TO THE BLACK SIX. THIS IS A WILDTYPE CELL F 100, CASTANEUS IS BEING EXPRESSED, AND EVERY CELL WE COUNT, WE SEE ALMOST ONLY ALL CASTANEUS RNA, BUT WE SEE A LITTLE BIT OF BLUE CORRESPONDING TO THE ALLELE THAT SHOULD BE OFF, WE KNOW THIS IS WILDTYPE AND IS OFF, WE DO A CONTROL, CASTANEUS BY CASTANEUS, NO BLACK SIX, WE SEE ABOUT THE SAME AMOUNT OF BLACK SIX SO THAT'S OUR BACKGROUND. THAT'S PROS HYBRIDIZATION. WHAT DOES THIS MUTANT LOOK LIKE? THIS MUTANT LOOKS LIKE THIS. THESE ARE AGAIN CELLS, AND IF YOU DO A LITTLE OUTTAKE WHAT YOU CAN SEE IS THERE'S TWO POPULATIONS OF CELLS. CELLS THAT EXPRESS BOTH MATERNAL AND PATERNAL ALLELE OF H19 ABSOLUTELY BIALLELICALLY AND CELLS THAT ARE LARGELY MONO ALLELIC OR WILD TYPE. SO WHAT DOES THAT MEAN? IT MEANS THAT WHAT WE'RE SEEING IS A MOSAIC MIXTURE OF CELLS THAT LOOK WILD TYPE AND CELLS THAT ARE COMPLETELY MUTANT AND BIALLELIC. OKAY. SO BECAUSE WE KNOW THAT SOME CELLS EXPRESS H19 MONO ALLELICALLY AND SOME EXPRESS BIALLELICALLY, WHAT DOES Igf2 LOOK LIKE IN THE SINGLE CELLS? SO REMEMBER WILDTYPE CONDITIONS YOU SHOULD ONLY SEE MATERNAL H19, AND PATERNAL Igf2, BUT WE KNOW THAT WE SEE CELLS THAT HAVE PATERNAL H19 OFF LIKE THE WILDTYPE AND PATERNAL H19 ON. SO EITHER THOSE CELLS WOULD HAVE Igf2 ON OR THOSE CELLS WOULD HAVE Igf2s ON, SO WHAT'S THE ANSWER? THE ANSWER IS ONLY WHEN YOU HAVE MONO ALLELIC H19 AND THERE'S LOTS OF EXAMPLES OF THAT DO WE EVER SEE Igf2. THE BIALLELIC CELLS NEVER EXPRESS Igf2. SO WHAT THAT MEANS IS THESE ENHANCERS CAN EITHER ENGAGE H19 OR Igf2 ON A SINGLE CHROMOSOME, THEY CAN'T ENGAGE THE TWO OF THEM AT THE SAME TIME. AND SO THAT'S REALLY IMPORTANT INFORMATION FOR UNDERSTANDING HOW THIS LOCUS WORKS. OKAY. HERE IS ANOTHER WAY THAT WE HAVE -- I CAN DEMONSTRATE THIS. THIS IS ACTUALLY OCCASIONALLY WE CAN SEE SITES OF TRANSCRIPTION IN THE NUCLEUS. HERE IS NUCLEUS AND CYTOPLASM. THERE'S TWO CHROMOSOMES. HERE IS THE GUIDE PROBE, WE SEE ONE SITE OF TRANSCRIPTION OF H19, WE KNOW THAT IT'S CASTANEUS BUT WE DON'T SEE ANY OF THE BLACK 6, MONO ALLELIC CELL, IT'S IN THIS CELL THAT WE SEE Igf2 EXPRESSION. IF WE HAVE A BIALLELIC CELL THAT HAS TWO SITES OF TRANSCRIPTION THIS GUY RIGHT HERE, THIS CASTANEUS, THIS THE GUY RIGHT HERE BLACK 6, WE SEE NO Igf2, CONVINCINGLY SHOWS US THAT YOU ONLY SEE Igf2 WHEN YOU DON'T HAVE H19 BEING EXPRESSED THERE THAT PARTICULAR CHROMOSOME. OKAY. SO IN THIS POPULATION OF CELLS FROM OUR MUTANT WE HAD 25% OF THE CELLS STILL EXPRESSED H19 MONO ALLELICALLY, WE DON'T REALLY UNDERSTAND WHY. 25 STILL FINE. 75 EXPRESS BIALLELICALLY, THOSE CELLS HAVE MORE BIALLELIC -- THE CELLS HAVE MORE TOTAL H19 THAN THE MONOALLELIC CELLS, BUT THE QUESTION IS THEN WHAT IS THE DIFFERENCE BETWEEN THAT CELL AND THOSE SET OF CELLS AND THAT SET OF CELLS? AND ONE OF THE THINGS THAT'S SORT OF INTRIGUING TO US IS IN THIS MUTANT POPULATION OF CELLS WE DID DNA METHYLATION ANALYSIS AND 25% OF THE STRANDS LOOK LIKE WILD TYPE, FULLY METHYLATED, SO EACH OF THESE IS A STRAND OF DNA AND IF IT'S FILLED IT MEANS IT'S METHYLATED, IF IT'S EMPTY IT'S UNMETHYLATED. AND SO WILD TYPE SHOWS YOU ALL METHYLATED. AND SO 25% OF THESE LOOK LIKE THIS. THE NEXT QUESTIONS THAT WE WANTED TO ASK, REALLY TWO QUESTIONS, HOW STABLE IS THAT ALLELIC EXPRESSION PATTERN? ONCE A CELL BECOMES MONOALLELIC OR BIALLELIC DOES IT STAY, AND WHAT IS THE MECHANISM FOR THIS ALLELIC PATTERN? TO ADDRESS THIS QUESTION WE DEVELOPED ANOTHER EXPERIMENT AND WHAT WE DECIDED TO DO, THESE ARE PRIMARY MOUSE EMBRYONIC FIBROBLASTS WE WERE LOOKING AT, AND THEY ONLY HAVE A FEW CELL DIVISIONS IN VITRO BUT WE PLATED THEM OUT DILUTELY ON A LAYER OF HUMAN FIBROBLAST, AND WE LET THE COLONIES GROW UP AND THEN WE IMAGED THEM SO WE COULD SEE COLONIES OF CELLS, AND WHAT WE SAW WAS WE THEN ASKED THE QUESTION DO ALL OF THE CELLS IN A GIVEN COLONY, ALL CORRESPONDING TO THE SAME STARTING CELL, THEY ARE ALL SORT OF OFFSPRING OF THE SAME CELL, WHAT DOES THEIR EXPRESSION PATTERN LOOK LIKE WHEN YOU IMAGE, AND WHAT IT LOOKS LIKE IS VIOLET COLONIES ARE BIALLELIC OR MONOALLELIC. SO WE SEE THAT ONCE A DECISION IS MADE, IT'S MAINTAINED AND STABLE. SO NOW THAT WE COULD GET THESE COLONIES, WE TRIED TO GROW THEM UP TO IMAGE THEM AND GET ENOUGH CELLS THAT WE COULD DO THE BISULFITE ON THEM AND WE FINALLY WERE ABLE TO DO THE IMAGING, FIXING THE IMAGING AND BISULFITE, AND WHAT WE SAW WAS AS WE EXPECTED WHEN A CELL LOOKED WILD TYPE OR MONOALLELIC IT HAD A NORMAL METHYLATION PATTERN, AND WHEN IT WAS ABNORMAL OR BIALLELIC IT HAD A LOSS OF METHYLATION. SO CONSISTENT WITH WHAT WE THOUGHT WAS THE BIALLELIC CELLS SHOWED THE ICR HAD A LOSS OF METHYLATION. OKAY. AND SO WE'VE NOW BEEN EXTENDING THIS SNP-FISH INTO TISSUES WHICH IS WHAT WE WANT TO DO TO LOOK AT COMPLEX POPULATIONS OF CELLS IN MOUSE TISSUES, OR MY FELLOW WOULD LIKE TO LOOK IN TISSUES IN SOME OF HER BECKWITH PATIENTS. WE CAN SEE THAT THERE ARE CLONAL EXAMPLES. THIS IS ACTUALLY CARDIAC TISSUE WHICH WAS EASIER TO DO THE IMAGING ON, AND YOU CAN SEE THAT THERE'S PATCHES OF CELLS THAT EITHER ARE BIALLELIC OR MONOALLELIC, SUGGESTING AGAIN THAT YOU HAVE A CELL THAT GOES THROUGH SOME SORT OF DECISION PROCESS, EITHER LOSES ALL ITS METHYLATION OR MAINTAINS METHYLATION, AND THEN THAT'S STABLE. OKAY. LET ME JUST SUMMARIZE WHAT I'VE SHOWN YOU IS WE NOW CAN IMAGE ALLELE-SPECIFICALLY USING POLYMORPHISMS AS A SINGLE CELL LEVEL BOTH IN CELLS AND IN TISSUES. WHAT I FORGOT TO TELL YOU IS THAT WHEN YOU QUANTIFY YOUR SIGNALS YOU GET FROM THE MICROSCOPIC IMAGING, THE SNP-FISH, IT'S IDENTICAL TO WHAT YOU SEE WHEN YOU DO BOTH CULTURES BY ITPCR, AND THAT WE END UP WITH EPIGENETIC MOSAIC MIXTURE IN THE LOSS OF IMPRINTING MUTANTS WAS STABLE, WHICH IS STABLE, SO WHAT DO WE THINK HAS GONE ON IN OUR MOUSE MUTANT? SO WE KNOW THAT WITH THAT PARTICULAR MUTATION WHEN IT COMES FROM THE FATHER IT HAS METHYLATION OF THE CpGs THAT ARE REMAINING, THAT CAN BE METHYLATED, AND THAT METHYLATION IS SORT OF PRESENT A LITTLE BIT IN THE BLASTOCYSTS BUT BEGINS TO BE LOST. THIS IS A PERIOD WHERE THERE'S A LOT OF REPROGRAMMING THAT GOES ON IN THE MAMMALIAN DEVELOPMENT BUT ONCE YOU GET POST IMPLANTATION, THE REPROGRAMMING IS DONE IN THIS SOMATIC TISSUE AND MAINTENANCE METHYL TRANSFERASE IS INDUCED AND IN PLACE, WE THINK EVERY CELL EITHER MADE A DECISION TO LOSE METHYLATION, BE BIALLELIC OR MAINTAIN AND BE MONOALLELIC, THAT'S STABALLY MAINTAINED. YOU CAN TREAT WITH METHYLATION INHIBITORS AND ACTUALLY KNOCK THE WILDTYPE-LOOKING CELL INTO A MUTANT-LOOKING CELL. SO THAT SORT OF IS THE IMAGING AND WE WOULD LIKE TO NOW USE THAT IN A BUNCH OF OTHER SYSTEMS BUT IN THE REMAINING FEW MINUTES I WANT TO TELL YOU ABOUT ANOTHER PROJECT WE'VE BEEN WORKING ON, THIS COLLABORATION I'VE BEEN DOING FOR MANY YEARS WITH RICHARD SCHULTZ AT PENN, AND THIS IS -- STUART MENTION THE THIS IN THE INTRODUCTION, ASSISTED REPRODUCTIVE TECHNOLOGIES WORK. IT'S BEEN KNOWN FOR A LONG TIME IN THE LARGE ANIMAL WORLD WHERE THEY OFTEN USE IN VITRO FERTILIZATION TO PROPAGATE STRAINS THAT THESE EMBRYOS ONCE THEY ARE TRANSFERRED BACK TO MOMS CAN END UP WITH LARGE OFFSPRING SYNDROME WHICH ENDS UP WITH A NUMBER OF PROBLEMS, PLACENTAL AND OTHER TYPES OF PROBLEMS. WE ALSO KNOW SOME OF THESE THINGS ARE ALSO -- HAVE BEEN OBSERVED IN THE MOUSE, BUT WHAT'S BEEN A LITTLE BIT LESS APPRECIATED UNTIL MORE RECENTLY IS THAT THERE IS SOME INCREASE OF PROBLEMS, STILL LOW, BUT SOME INCREASE IN PROBLEMS THAT ARE ASSOCIATED WITH ASSISTED REPRODUCTION, AND WE KNOW THAT THERE'S PRE-TERM LABOR, PRE-TERM GROWTH, LOW BIRTH WEIGHT, SOME INCREASING CONGENITAL DISORDERS, THERE ARE OTHER PROBLEMS THAT ARE VERY CONTROVERSIAL IN THE LITERATURE ABOUT WHETHER THERE'S AUTISM OR OTHER THINGS LIKE THAT AND I THINK THERE'S A REALLY IMPORTANT STUDY THAT WAS DONE BY NICHD THAT JUST CAME OUT THAT SORT OF SUGGESTED THAT THERE WASN'T AN INCREASE IN AUTISM IN ART, WHICH IS IMPORTANT, BUT WHAT WE'VE BEEN INTERESTED IN WAS THE OBSERVATION THAT THERE SEEMS TO BE AN INCREASE IN IMPRINTING DISORDERS WHICH REMEMBER ARE STILL VERY RARE. THEY ARE STILL, YOU KNOW, ABOUT ONE IN 10,000 SO YOU NEED INCREDIBLY LARGE COHORTS TO REALLY SEE AN INCREASE. SOME OF THE STUDIES THAT HAVE BEEN DONE, FOR INSTANCE WITH BECKWITH-WIEDEMANN SUGGEST A SIX-FOLD RELATIVE RISK IN SEEING BECKWITH IN ASSISTED REPRODUCTION IN CHILDREN. SO WHEN I TALK ABOUT -- LET'S GO OVER REPRODUCTION, PARTICULARLY IN VITRO FERTILIZATION. WHAT HAPPENS HERE, YOU HAVE A PRODUCTION STIMULATED BY HORMONES, THE EGGS ARE RETRIEVED, THEN COMBINED WITH SPERM IN A PETRI DISH, ALLOWED TO FERTILIZE, OR IN SOME CASES SPERM ARE INJECTED INTO THE EGGS, THEY THEN WILL GROW FOR A COUPLE DIVISIONS IN THE DISH AND THEN TRANSFERRED BACK TO MOM, SO YOU HAVE SUPER OVULATION, EMBRYO CULTURE AND EMBRYO TRANSFER. WHAT'S REALLY IMPORTANT ABOUT THIS IS THESE PARTICULAR PROCEDURES TAKE PLACE AT A REALLY SENSITIVE TIME IN DEVELOPMENT, WHEN THE GENOMES ARE BEING REPROGRAMMED, SO FOR EXAMPLE SHOWN HERE IS -- THIS IS DNA METHYLATION, INCREASING DNA METHYLATION, SORT OF GENOME-WIDE, WEEKEND YOU SEE PGC, YOU LOSE METHYLATION, PROGRAMMING, IT'S REACQUIRED, AFTER FERTILIZATION IT'S LOST AGAIN, IF THE GENOME IS BEING REPROGRAMMED AND REACQUIRED, AND SUPER OVULATION TAKES PLACE WHEN OOCYTES ARE ACQUIRING, DURING MATURE IS A, ATION, GOING FROM SPECIALIZED EGGS AND SPERM INTO PLURIPOTENT CELLS AND DETERMINED INTO CELLS GOING IN VARIOUS LINEAGES, SO YOU SEE ALL THIS DNA METHYLATION REPROGRAMMING, IMPORTANTLY IMPRINTED GENES AFTER FERTILIZATION HAVE TO MAINTAIN THEIR IDENTITY AND DNA METHYLATION, THEY MAINTAIN BY MECHANISMS WE DON'T FULLY UNDERSTAND AT THIS TIME. SO WE AND OTHERS HAVE DONE LOTS OF EXPERIMENTS USING THE MOUSE MODEL TO ADDRESS THESE QUESTIONS, ULTIMATELY WHY THERE ARE INCREASED RISK OF EPIGENETIC PERTURBATIONS AND IMPRINTING DISORDERS IN ART, CHILDREN CON CONCEIVED IN ART, WITH THE ULTIMATE GOAL OF MAKING IT SAFER AND WHICH PROCEEDURES ARE CONTRIBUTING TO THIS. THERE WAS A LOT OF IMPRINTING AND LOSS IN DNA METHYLATION IN THE MOUSE, IMPRINTING CONTROL REGION, AFTER EMBRYO CULTURE. THERE WAS LOSS OF METHYLATION, ASSOCIATED WITH SUPER OVULATION. A FORMER FELLOW IN THE LAB SHOWED LOSS OF IMPRINTING, LOSS OF METHYLATION THAT COULD BE ASSOCIATED WITH EMBRYO TRANSFER, TAKING THE EMBRYO OUT OF ONE MOUSE, TAKE IT OUT, PUT IT INTO ANOTHER MOUSE, SO AN ENVIRONMENTAL PERTURBATION. AND WE SEE -- WE RECENTLY SHOWED YOU HAD MORE SEVERE LOSS OF IMPRINTING AND LOSS OF METHYLATION WHEN THE ENTIRE IVF PROTOCOL IS USED, BUT THAT WAS REALLY FOCUSED IN THE PLACENTA WHERE YOU SAW THE GREATEST CHANGES. SO THE QUESTIONS THAT WE ASKED, MOST RECENT, WITH TWO FELLOWS IN MY LAB, ERIC AND LISA, CAN WE ASSOCIATE THESE SPECIFIC PROCEDURES THAT ASSISTED REPRODUCTION, IVF, IN VITRO FERTILIZATION, WITH VARIOUS PATHOLOGIES, SPECIFICALLY I'M GOING TO FOCUS ON THE PLACENTA IN THE LAST FEW MINUTES. WHAT LISA AND ERIC DID WAS THEY DESIGNED AN EXPERIMENT WHERE WE COULD DISTINGUISH THE VARIOUS PROCEDURES SO WE FIRST HAVE NATURAL MATING, MICE NATURALLY MATE, AND THEN WHAT WE'RE GOING TO DO WE LET THEM GESTAT TO TERM, SACRIFICED THE MOUSE, REMOVED THE EMBRYO AND PLACENTA. MORE RECENTLY CESEAREAN DELIVER THE MICE, FOSTER THEM, TAKE THE PLACENTA, ASSOCIATE THE PLACENTA WITH PHENOTYPE OF THE OFFSPRING. I'M GOING TO TALK ABOUT TERM PLACENTA. THAT'S THE NATURAL SITUATION. AND THEN WE HAVE THIS CONTROL WHERE THERE'S JUST PURELY EMBRYO TRANSFER, SO THE MICE -- NO SUPER OVULATION, MICE MATE, REMOVE EMBRYO, PUT IT BACK IN, FOSTER MOM GESTATES, TRANSFER BACK, SUPER OVULATION, TRANSFER, SUPER OVULATE THE MOM, LET THEM MATE. TAKE THE EMBRYOS OUT, TRANSFER TO FOSTER MOM AND THE WHOLE IVF PROCEDURE OF SUPER OVULATION CULTURE, IVF CULTURE, TRANSFERRED, OKAY. AND SO I'LL SUMMARIZE THE FINDINGS ON THE NEXT FEW SLIDES. SO THE FIRST THING WE LOOKED AT IS PLACENTA, THIS IS PLACENTA WEIGHT. SO WHAT IS SHOWN IS EACH OF THESE GRAPHS WILL BE THE SAME WITH THE NATURAL, EMBRYO TRANSFER, SUPER OVULATION, EMBRYO TRANSFER AND IVF, AND WHAT WE SEE IS THAT EITHER BY PLACENTAL WEIGHT OR PLACENTAL TO FETAL WEIGHT WE SEE ALL OF THESE CONDITIONS RESULT IN AN INCREASE IN WEIGHT RELATIVE TO THE EMBRYO. SO JUST EMBRYO TRANSFER ALONE, AND THEN YOU SEE SORT OF IT WORSEN WHEN YOU USE THE WHOLE PROCEDURE. AND SO HERE IS AN EXAMPLE. THESE ARE H AND E STAINED PLACENTAS. HERE IS NATURAL, HERE IS IVF. IT'S PRETTY OBVIOUS THEY ARE LARGER. YOU DON'T NEED A LOT OF DEGREES TO SEE THAT. EVEN I CAN TELL THAT. AND SO THEN WHAT MY -- WHAT LISA AND ERIC DID WAS THEY STAINED AND THEY LOOKED FOR THE JUNCTIONAL VERSUS LABYRINTH ZONE, THIS IS PSEUDOCOLORED TO SHOW YOU HERE IS HOW MUCH YOU EXPECT TO SEE OF THE JUNCTIONAL ZONE VERSUS LABYRINTH, WHERE YOU HAVE EXCHANGE BETWEEN MOM AND FETUS AND JUNCTIONAL HAS SOME ENDOCRINE-SECRETING CELLS, ET CETERA, A VERY BASIC POINT. AND YOU CAN SEE THAT AS YOU GO ON, ESPECIALLY ONCE YOU GET TO IVF, THE JUNCTIONAL ZONE RELATIVE TO THE LABYRINTH IS GREATLY EXPANDED, AND WHEN YOU DO THE MEASUREMENTS, AND THIS IS ALL DONE BLINDED, THIS IS LIKE ONE OF THE MOST IMPORTANT EXPERIMENTS TO DO BLINDED THAT I'VE EVER COME UP WITH, WE ACTUALLY HAVE A FLEET OF UNDERGRADUATES IN THE LAB THAT WE HAND THESE TWO AND HAVE THEM DO THE -- LOOK AT THE AREA. AND SO WHAT WE SEE IS THAT NATURAL AND EMBRYO TRANSFER DON'T LOOK STATISTICALLY DIFFERENT BUT WHENCE YOU GET TO SUPER OVULATION AND TRANSFER AND IVF YOU SEE ENHANCED JUNCTIONAL TO LABYRINTH RATIO. SO THEN WE LOOKED AT SOME MOLECULAR BIOLOGY OF THESE PLACENTAS, AND ONE OF THE THINGS WE LOOKED AT WAS DNA METHYLATION OF THESE IMPRINTING CONTROL REGIONS, AND THIS IS DONE IN BULK, YOU HAVE A METHYLATED ALLELE AND UNMETHYLATED ALLELE. NORMALLY YOU WOULD SEE 50% METHYLATION IF YOU'RE NOT DOING AN ALLELE-SPECIFICALLY. AND WHAT YOU CAN SEE IS FOR THESE THREE IMPRINTING CONTROL REGIONS, THERE'S REDUCED METHYLATION THAT'S STATISTICALLY SIGNIFICANT, ONLY IN THE IVF, AND SIMILARLY WHEN WE LOOK AT GENOME-WIDE METHYLATION WORKS ARE WE SEE REDUCED, ONLY IVF SHOW METHYLATION CHANGES. I'M GOING TO SHOW YOU REAL FAST WHAT DO THE FETUSES LOOK LIKE AT THIS POINT. THIS IS EXPRESSION, ALLELE-SPECIFIC EXPRESSION, AND YOU SEE A COUPLE FETUSES THAT HAVE SOME CELLS THAT LOOK BIALLELIC, THIS IS WHAT WE LIKE TO DO OUR ALLELE-SPECIFIC SNP-FISH TO SEE HOW MANY CELLS, CERTAIN TYPES OF CELLS, BUT YOU DO SEE STATISTICALLY MORE EXPRESSION FROM THE NORMALLY REPRESSED ALLELE, ALLELE SHOULD BE OFF, SO AT THIS BASAL LEVEL IN THE IVF VERSUS THE NATURAL, BUT ACTUALLY IF YOU LOOK AT THE ICR METHYLATION, THE METHYLATION IS NOT REALLY THAT BAD, SO THERE'S PROBABLY JUST A FEW CELLS IN THAT POPULATION ARE EXPRESSING THE NORMALLY REPRESSING HILATE. IN THE CASE OF H19 WHEN WE HAD A MOUSE WITH REDUCED METHYLATION, OR TISSUE THAT HAD REDUCED METHYLATION OR BIALLELIC EXPRESSION, THAT WE WOULD SEE IT IN THE BRAIN AS WELL AS IN THE LIVER, SO THE MISTAKE HAPPENED EARLY IN DEVELOPMENT AND WAS MAINTAINED. HOWEVER, THIS IS PLACENTA, AND PLACENTA SHOWS MUCH WORSE PHENOTYPE THAN WHAT YOU SEE IN THE MOUSE. MOST OF THE TIME OF THE FETUS LOOKS FINE, BUT IN THIS CASE THE PLACENTA IS ADVERSELY AFFECTED. WHAT THIS TELLS US IS THE MORE WE MANIPULATE, THE MORE MANIPULATIONS WE INTRODUCE, THE MORE SEVERE THE PLACENTAL AND IN THE END EMBRYONIC PHENOTYPE, EMBRYO TRANSFER, WHEN WE INTRODUCE OVULATION WE SEE JUNCTIONAL ZONE OVERGROWTH AND WHEN WE INTRODUCE ALL OF THE PROCEDURES IN IV, IF WE SEE PERTURBATIONS IN DNA METHYLATION AND GENE EXPRESSION, MUCH MORE SERIOUSLY. THIS SHOWS US THAT EACH OF THESE PROCEDURES CONTRIBUTES A LITTLE BIT AND SO EACH ONE OF THEM MAY HAVE SOME KIND OF -- REQUIRE SOME KIND OF ADJUSTMENT, AND WE ALREADY KNOW NOW THAT FOR EXAMPLE FOR IN TERMS OF SUPER OVULATION, WOMEN ARE GIVEN MUCH LOWER DOSES OF HORMONES WHEN BEING SUPER OVULATED WHICH IS IMPORTANT IN BEING OPTIMIZED, THIS IS A NICE SYSTEM FOR STUDYING CHANGES AND PROCEDURES IN ASSISTED REPRODUCTION. SO WHAT WE'RE MOVING ON TO DO IS TO FIGURE OUT IF THESE MORPHOLOGICAL CHANGES IN PLACENTA CAN BE ASSOCIATED WITH ABERRANT OUTCOMES LATER ON AND WE DEVELOPED A VERY NICE STRATEGY TO C-SECTION THESE MICE AND TRANSFER AND MARK THE MICE WE CAN TRACK BACK THE PLACENTA AND STUDY THEM AND WE'RE ALSO PROFILING ALL THE TISSUES TO FIGURE OUT EXTENT OF CHANGES AND WE REALLY WANT TO KNOW WHY WE SEE LOSS OF METHYLATION BECAUSE WE REALLY BELIEVE THAT REALLY THE CAUSE OF A LOT OF PROBLEMS THAT WE HAVE OBSERVED IN THESE MICE. SO FINALLY I JUST WANT TO ACKNOWLEDGE THE PEOPLE WHO DID THE WORK. THIS IS A NICE SUNNY DAY AT PENN WITH OUR LEADER, FORMER LEADER, BENJAMIN FRANKLIN, SHOWN HERE, THE PEOPLE WHO CONTRIBUTED TO THE WORK THAT I DESCRIBED TODAY IN PURPLE, TOGETHER WITH OUR COLLABORATORS RAJ AND THE LAB FOR THE FISH AND LONG STANDING COLLABORATION WITH RICHARD SCHULTZ AND PEOPLE IN HIS LABORATORY AND FORMER MEMBERS OF MY OWN LAB, AND PROBABLY WOULDN'T BE STANDING HERE TODAY IF IT WASN'T FOR THE FACT THAT I HAVE VERY GENEROUS SUPPORT FROM THE NIH AS WELL AS SOME OTHER ORGANIZATIONS, AND SO I REALLY WOULD LIKE TO THANK YOU FOR YOUR ATTENTION AND I THINK I CAN TAKE QUESTIONS. OKAY, THANK YOU. [APPLAUSE] ANY QUESTIONS? >> YEAH, WHEN YOU'RE TALKING ABOUT THE QUESTION OF WHY THE PLACENTA WOULD BE THE MOST EFFECTIVE, AFFECTED, IT WOULD SEEM ALMOST OBVIOUS BECAUSE ONE OF THE FIRST EVENTS THAT HAPPENS WHEN YOU WOULD TRANSFER A DEVELOPED EMBRYO OR ZYGOTE TO A SURROGATE UTERUS WOULD BE IMPLANTATION AND SEPARATION OF THE EMBRYO AND PLACENTA. THEREFORE IT'S MISSING WHATEVER INTERNAL STIMULUS, EPIGENETIC STIMULUS, WHILE IT'S IN PROCESS OF IVF. YOU KNOW WHAT I'M SEEING? I'M SAYING IN OTHER WORDS IF IN THE PROCESS OF INTRAWHATEVER, WHEN YOU'RE DOING ARTIFICIAL, ALL THESE THING ON THE OUTSIDE BEFORE YOU PUT IT IN THERE, IT SHOULD BE HAVING THE BENEFIT OF BEING WITHIN THE MATERNAL ENVIRONMENT. >> I MEAN, I THINK THAT'S AN IMPORTANT QUESTION. THERE'S ALSO THE QUESTION OF WHETHER THERE'S, YOU KNOW, GENETIC DIFFERENCES BETWEEN MOTHER AND OFFSPRING BUT THAT'S STILL THE -- I MEAN STILL AN IMPORTANT QUESTION OF WHY THAT HAPPENS, AND IF THERE'S A WAY TO REMEDIATE THE SITUATION BECAUSE YOU CAN'T, IN IVF, YOU HAVE NO RECOURSE. YOU HAVE TO TRANSFER IT TO ANOTHER MOM. IT'S HAPPENING IN PETRI DISHES, GOING BACK INTO THE MOM, NOT SITTING THERE THE WHOLE TIME. SO I THINK THAT'S AN IMPORTANT QUESTION. >> OKAY. I THINK THAT IT WOULD BE ODD IF THE PLACENTA WAS NOT THE THING THAT WOULD BE MOST AFFECTED. >> I THINK THAT A LOT OF WHAT WE FOUND WHEN WE HAVE BEEN DOING A LOT OF MANIPULATIONS IN ENDOCRINE DISRUPTORS, ANY ADVERSE ENVIRONMENT SEEMS TO AFFECT THE PLACENTA, MORE THAN YOU WOULD HAVE UNDERSTOOD, SO I THINK IN SOME OF OUR ENDOCRINE DISRUPTOR WORK WHERE THERE'S NO TAKING OUT OR PUTTING BACK IN, IT'S JUST AN EXPOSURE, WE SEE SIMILAR PLACENTAL PHENOTYPES SO THERE'S SOMETHING ELSE GOING ON IN ADDITION TO WHAT YOU'VE BROUGHT UP. SO I THINK THE PLACENTA IS VERY, VERY SENSITIVE TO ANY ADVERSE ENVIRONMENTAL INSULT AND THAT I THINK MECHANISTICALLY TRYING TO UNDERSTAND WHAT IT IS, WHEN WE LOOK ACROSS OUR DIFFERENT SYSTEMS, IS REALLY AN IMPORTANT QUESTION THAT WE'RE TRYING TO ADDRESS. >> JUST A POINT, I HAD DONE A FELLOWSHIP IN PERINATAL, NEONATAL PATHOLOGY IN HUMANS, QUITE A FEW AUTOPSY ON ABORTIONS, YOU KNOW, WE WOULD ALWAYS INCLUDE IN THE AUTOPSY A PLACENTA AND A FETUS, IN OTHER WORDS THAT'S PART OF THE BASIC ORGAN. AND, YOU KNOW, THAT'S KIND OF MY CLASSIC -- THE FIRST THING WE LOOK AT IN THE PLACENTA, LEADING ME TO THE CONCLUSION BASED ON EXPERIENCE ALSO. >> RIGHT. BUT ALSO THE OTHER SITUATION, THE OTHER QUESTION, WHY YOU SEE INCREASING PROCEDURES GIVE YOU MORE ADVERSE PATHOLOGY TOO, SO THAT'S ANOTHER QUESTION OF WHAT WE'RE TRYING TO UNDERSTAND ALSO. >> ALL RIGHT. THANK YOU VERY MUCH. >> UH-HUH. OTHER QUESTIONS? IN A BIG ROOM PEOPLE ARE EMBARRASSED TO ASK QUESTIONS. YOU CAN ASK A QUESTION, YEAH. >> (INAUDIBLE). >> WE HAVEN'T DONE THAT YET. I THINK THAT'S A QUESTION THAT PEOPLE WANT TO KNOW, DO -- SOME PEOPLE HAVE DONE SOME EXPERIMENTS WHERE THEY SAY ARE THE GERM CELLS AFFECTED, AND I WOULD SAY THAT IN THOSE MICE NOW, I CAN'T DO ANYTHING WITH THEM, THAT HAD LOWER METHYLATION IN MULTIPLE TISSUES, YOU WOULD EXPECT THAT THEIR GERMLINES WOULD HAVE A POTENTIALLY SIMILAR SORT OF DEFECT, IF IT COULD HAVE A DEFECT, IF THERE'S A QUESTION OF ABERRANT METHYLATION AND REMETHYLATION, AND SO WE NEED TO DO A LARGER STUDY AND WE'RE GOING TO FIRST LOOK AND SEE WHETHER WE'RE TRYING TO IDENTIFY PHENOTYPES, CERTAINLY ONE IS PLACENTAL PHENOTYPE, OTHER PHENOTYPES THAT WE CAN LOOK INTO IN THE NEXT GENERATION TO SEE IF ANY THAT TRANSFERRED YET BECAUSE THE ENDOCRINE DISRUPTOR WORK WE HAVE PHENOTYPES IN THE THIRD GENERATION WHICH SAYS THEY ARE TRANSGENERATIONAL SO WE LIKE TO SEE OTHER ENVIRONMENTAL INSULTS CAN BE TRANSMITTED. I HOPE NOT BUT ... UH-HUH? >> YOUR MODEL IN THE FIRST PART PREDICTS THAT THE SEVERITY OF A BECKWITH PATIENT WILL BE ABOUT THE TIMING OF EPIMUTATION, IF YOU LOOK AT PATIENTS MORE SEVERE YOU WOULD EXPECTS MORE BIALLELICALLY EXPRESSING CELLS? >> I REALLY BELIEVE THAT THE MORE SEVERE ONES IT HAPPENS EARLIER. AND BUT IT'S REALLY DIFFICULT ACCESSING THE TISSUES. I'VE BEEN TOLD THAT THERE HAVE BEEN SOME PATIENTS THAT DON'T LOOK VERY SEVERE BUT THEY WILL END UP WITH A KIDNEY TUMOR, AND BY BLOOD THEY HAVE A LOW LEVEL OF EFFECT, AND THEY GO IN AND TAKE OUT THE KIDNEY TUMOR AND ADJACENT TISSUE AND THEY SAY THERE'S MUCH WORTH EPIGENETIC PROFILE THAT'S ASSOCIATED WITH IT SO IT'S NOT NECESSARILY EASY TO DETECT BUT IN CASES WHERE THEY HAVE BEEN ABLE TO LOOK, THAT SEEMS TO BE CONSISTENT AND WE COULD SUGGEST THAT THOSE, THAT I THINK THOSE BECKWITH EPIMUTATIONS ARE MAINTENANCE PROBLEMS, AND SOMETIMES IT HAPPENS EARLIER, AND IT RECOVERS, AND SOMETIMES IT DOESN'T. THAT'S WHY WE REALLY ARE TRYING TO GENERATE A MOUSE MODEL TO BE ABLE TO STUDY THAT. WE'VE SO FAR BEEN UNSUCCESSFUL. >> ANOTHER QUESTION ABOUT THE SECOND PART. SO IT SEEMS LIKE IN SOME CASES THE PHENOTYPE ON ALLELIC EXPRESSION IS MORE DRAMATIC THAN THE DNA METHYLATION PHENOTYPE. >> YEAH. >> I MEAN HAVE YOU LOOKED AT OR IT'S MUCH HARDER TO I ASSESS BUT THERE'S CHROMATIN MARKERS AFFECTED BY MARKERS, IT MAY NOT BE DNA METHYLATION. >> THERE'S NEVER ENOUGH TIME TO TALK ABOUT THAT. WHEN YOU LOOK AT THE EXPRESSION, SEEMS TO BE ALL KINDS OF EXPRESSION FROM THE REPRESSED ALLELE, AND I WOULD ARGUE THAT IT JUST TAKES A COUPLE CELLS TO BE BLASTING EXPRESSION TO ALL OF A SUDDEN SEE WHAT LOOKS REALLY BAD SO WHEN YOU'RE ASSAYING A BIG POPULATION OF CELLS YOU'RE ASSAYING CELLS EXPRESS GENE BUT LOOKING AT DNA METHYLATION EVERY SINGLE NUCLEUS GIVES YOU A SIGNAL. PREDICTION, IF YOU USE THE SNP-FISH ASSAY WHICH I LOVE, WE'RE CLOSE TO THAT, I WOULD PREDICT WHERE YOU SEE THOSE BLASTING THING BUT METHYLATION LOOKS FINE MY PREDICTION IS IS FEW CELLS ARE COMPRESSING BIALLELIC BUT IF MORE ARE WE HAVE TO LOOK AT CHROMATIN AND OTHER THINGS SUGGESTING YOU'RE RIGHT THE DNA METHYLATION IS NOT AT PREDICTIVE AS YOU SUGGEST, IMPORTANT POINT. I DIDN'T TELL HIM TO ASK THAT QUESTION. >> (INAUDIBLE). >> THANKS. [APPLAUSE]