Download - Dima Chaar Senior Thesis presentation
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GeneticFateMappingofHoxa5-ExpressingCells
inMouseEmbryoSomites
Dima ChaarLab Mentor: Professor Mansfield
Barnard College, Columbia UniversityProfessor Romeo
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WhatareHox genes?• Hox genesencodeTranscriptionFactors
• Transcriptionfactorsareproteinsthatbindandcontroltheactivityofothergenes
• ThecombinationofHox genesexpressedinembryonicmusculoskeletalandneuraltissuesspecifiesregionalidentityofthosetissuesalonghead-to-tailaxis
• Hoxmutationsleadtohomeotictransformations
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RoleofHox genesinDevelopment• Duringdevelopment,
• Mostofthemusculoskeletalsystemarisesfromsomites
• Thebrainandspinalcordarisefromtheneuraltube
• Somitescompartmentalizeandtheirderivativesdifferentiate
• Hox genesareexpressedinthesomitesandneuraltube,wheretheyregulateregionalidentity
Dermomyotomeà• Dermis• Muscle• MuscleConnectiveTissue
• Adipocytes
Sclerotomeà• Chondrocytes(cartilage)
• Perichondrium• Ligaments• Tendons
NeuralTubeà• Neurons
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Myfocus:Hoxa5• Hoxa5 expression occurs in the
cervical-to-thoracic transition in somites
• Hoxa5 is required for regionalization of the musculoskeletal and nervous systems in the same area
• I will be focusing on Hoxa5’s role in somite differentiation
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•Weknow:
• WhereHoxa5 isexpressed
• WhatrolesHoxa5 performsinvertebraldevelopmentfromstudiesof
loss-of-functionmutations
•Wedon’tknow:
• HowHoxa5 playsitscrucialroleinvertebraldevelopment
• WhichtissuesarederivedfromHoxa5-expressingcellsandwhenisHoxa5
expressionnecessary?
• Transcriptionaltargets
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• MyresearchfocusedondeterminingthecellfatesofHoxa5-expressingcellsandtheirdescendantsatdifferenttimepoints
•ResearchQuestions1. WhichtissuetypesdoHoxa5 descendantsbecome?2. IsthereaparticularspatialdistributionofHoxa5
descendantswithinatissue?3. HowdotheseHoxa5-expressingcellsbehave
differentlythanothers?
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HowdidIfatemap?
Method:UsedinducibleCre-ERT2linetopermanentlylabelHoxa5-expressingcellsandtheirdescendants
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Nucleus
ERT2
CodingSequence(CDS)
Hoxa5promoter
DNA
CreRecombinase-ERT2
Tamoxifenbinds
Cre-ERT2receptor
Tamoxifen
STOPCassette
LoxP LoxP
TomatoCDS
TomatoCDSConstitutivepromoter
DNA
DNA
Figure1:TheInducibleCre-ERT2model
Cytoplasm
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Cre-ERT2allowstemporalfatemappingofHoxa5-expressingcells
EmbryonicStages(days) e7.0e8.5 e9.5e10.5e11.5e12.5e13.5e14.5e15.5
Hoxa5expressionbegins
TamoxifenInjection
Hoxa5-expressingcellslabelledwithCre-ERT2
HarvestHoxa5 EndogenousExpression
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DetermineoptimaldoseofTamoxifenforCreinductioninmice
IdentifyHoxa5descendantsat3
differenttimepoints
Methods:Timeline
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Experiment1: Generate embryosinwhichtheHoxa5 -expressingcellsandtheirdescendantsarelabelledwithTdtomato
InjectTamoxifenatspecificdose
(9,12or15mg/40mg)
Harvestandembedembryos
inOCT
Sectionwithcryostat
Immuno-histochemistry
MicroscopyandPhotography
Ate8.5
Ate14.5
ChooseTamoxifendosewith• AdequateTdtomatolabeling
• Embryossurvive
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Generating embryosinwhichtheHoxa5-expressingcellsandtheirdescendantsarelabelledwithTdtomato
XHoxa-5Cre/+Male Tdtomato /Tdtomato Female
Hoxa-5Cre/Tdtomato embryo
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Generate embryosinwhichtheHoxa5 -expressingcellsandtheirdescendantsarelabelledwithTdtomato
InjectTamoxifenatspecificdose
(9,12or15mg/40mg)
Harvestandembedembryos
inOCT
Sectionwithcryostat
Immuno-histochemistry
MicroscopyandPhotography
Ate8.5
Ate14.5
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• Tamoxifenwasintraperitoneallyinjectedatdifferent trialdoses(9,12or15mg/40mgofbody weight)ate8.5
•Ate14.5,embryoswereharvestedandembeddedinOCT
• Embryosweresectionedusingacryostat
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Generate embryosinwhichtheHoxa5 -expressingcellsandtheirdescendantsarelabelledwithTdtomato
InjectTamoxifenatspecificdose
(9,12or15mg/40mg)
Harvestandembedembryos
inOCT
Sectionwithcryostat
Immuno-histochemistry
MicroscopyandPhotography
Ate8.5
Ate14.5
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• Immunohistochemistryformuscleactin
•MicroscopywasperformedtoimagetheTdtomato inHoxa5
descendants
•Determinewhichdoseproducedoptimalflorescence
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Immunohistochemistry,MicroscopyandPhotography
Sectionofe9.5injectedembryowithoptimaltamoxifendoseof9mg/40mgofbodyweight
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Experiment2:FateMapHoxa5descendantsinsomitesfrome9.5,e12.5ande13.5
Generate embryosinwhichtheHoxa5 -expressingcellsandtheirdescendantsarelabelledwithTdtomato
Injectoptimal doseof9mg/40gofbody
weight
HarvestingandOCTembeddingof
embryos
Sectioningwithcryostat
Immuno-histochemistry
MicroscopyandPhotography
Ate9.5,e12.5&e13.5
Ate14.5ore15.5
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Method:Timeline
EmbryonicStages(days) e7.0e8.5 e9.5e10.5e11.5e12.5e13.5e14.5e15.5
Hoxa5expressionbegins
TamoxifenInjection
Hoxa5- expressingcellslabelledwithCre ERT2
TamoxifenInjection
TamoxifenInjection
Harvest HarvestHarvestHoxa5 EndogenousExpression
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Results:LabelingHoxa5 expressingcellsate9.5-10.5
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MuscleActinA5descendants
MuscleActinA5descendants
MuscleActinA5descendants
MuscleActinA5descendants
MuscleActin
MuscleActinA5descendants
MuscleActinA5descendants
e9.5 e9.5e9.5
e12.5 e12.5 e13.5 e13.5
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e9.5 e12.5 e13.5
MuscleActinA5descendants
MuscleActinA5descendants
MuscleActinA5descendants
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e9.5 e12.5 e13.5
MuscleActinA5descendants
MuscleActinA5descendants
MuscleActinA5descendants
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Summary:Hoxa5-expressingcellsshowtissue-typerestriction
• TransgenicCre lineandtheCre-ERT2linerevealedtissue-specificrestrictionofcellsthat expressHoxa5
• MyCre-ERT2resultssuggestthatHoxa5expressingcellsgiverisetochondrocytesinatime-dependentmanner
Dermomyotomeà• Dermis• Muscle• MuscleConnectiveTissue
• Adipocytes
Sclerotomeà• Chondrocytes• Perichondrium• Ligaments• Tendons
✓
✓
✓
✓
✓= Hoxa5 descendantscontributetothesetissues
NeuralTubeà• Neurons✓
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TissueTypes e9.5 e12.5 e13.5
Chondrocytes No Yes Yes
Perichondria Yes Yes Yes
MuscleFibroblasts Yes Yes Yes
Dermis Yes Yes Yes
Adipocytes Yes Yes Yes
Neurons Yes Yes Yes
Tamoxifen Injection Time Points
• Hoxa5-expressingcellsgaverisetodermis,perichondriaandadipocytesate9.5-13.5
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TissueTypes e9.5 e12.5 e13.5
Chondrocytes No Yes Yes
Perichondria Yes Yes Yes
MuscleFibroblasts Yes Yes Yes
Dermis Yes Yes Yes
Adipocytes Yes Yes Yes
Neurons Yes Yes Yes
Tamoxifen Injection Time Points
• Theabsenceofdescendantsinthevertebralbodyate9.5suggeststhatchondrocytesdonotexpressHoxa5 untilaftere10.5.
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Conclusion
•Hoxa5 patternsregionalidentitybyactingon
asubsetoftissuetypes
•Hoxa5-expressingcellscontributeto
chondrocytesinatemporal-specificmanner
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FutureDirections
Refinetimepointinvestigationsbyfatemappingateachgestationdaybetweene9.5ande12.5
IdentifytranscriptionaltargetsofHoxa5inthetissuesandtimepointsthatwehavecharacterizedashavingHoxa5descendants.
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Implications• StudyingtheroleofHoxa5 iscrucialtounderstanding
• Complexities behindembryologicaldevelopment
• Howearlymutationscouldleadtophysicalabnormalities
andhealthdefects
• Withthisinformation,future doctorscouldpotentially
treatorpreventphysicalabnormalitiesusinggene
therapy
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• NationalScienceFoundation• HowardHughesSciencePipelineProject• MyLabmentor,ProfessorMansfield• Mythesismentor,ProfessorRomeo• FellowN&Bmajors• PastandPresentStudentsoftheMansfieldLab• Biology&NeuroscienceDepts.• FriendsandFamily!J