Dima Chaar Senior Thesis presentation

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  • GeneticFateMappingofHoxa5-ExpressingCells

    inMouseEmbryoSomites

    Dima ChaarLab Mentor: Professor Mansfield

    Barnard College, Columbia UniversityProfessor Romeo

  • WhatareHox genes? Hox genesencodeTranscriptionFactors

    Transcriptionfactorsareproteinsthatbindandcontroltheactivityofothergenes

    ThecombinationofHox genesexpressedinembryonicmusculoskeletalandneuraltissuesspecifiesregionalidentityofthosetissuesalonghead-to-tailaxis

    Hoxmutationsleadtohomeotictransformations

  • RoleofHox genesinDevelopment Duringdevelopment,

    Mostofthemusculoskeletalsystemarisesfromsomites

    Thebrainandspinalcordarisefromtheneuraltube

    Somitescompartmentalizeandtheirderivativesdifferentiate

    Hox genesareexpressedinthesomitesandneuraltube,wheretheyregulateregionalidentity

    Dermomyotome Dermis Muscle MuscleConnectiveTissue

    Adipocytes

    Sclerotome Chondrocytes(cartilage)

    Perichondrium Ligaments Tendons

    NeuralTube Neurons

  • 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 Hoxa5s role in somite differentiation

  • Weknow: WhereHoxa5 isexpressed

    WhatrolesHoxa5 performsinvertebraldevelopmentfromstudiesofloss-of-functionmutations

    Wedontknow: HowHoxa5 playsitscrucialroleinvertebraldevelopment

    WhichtissuesarederivedfromHoxa5-expressingcellsandwhenisHoxa5expressionnecessary?

    Transcriptionaltargets

  • MyresearchfocusedondeterminingthecellfatesofHoxa5-expressingcellsandtheirdescendantsatdifferenttimepoints

    ResearchQuestions1. WhichtissuetypesdoHoxa5 descendantsbecome?2. IsthereaparticularspatialdistributionofHoxa5

    descendantswithinatissue?3. HowdotheseHoxa5-expressingcellsbehave

    differentlythanothers?

  • HowdidIfatemap?

    Method:UsedinducibleCre-ERT2linetopermanentlylabelHoxa5-expressingcellsandtheirdescendants

  • Nucleus

    ERT2

    CodingSequence(CDS)

    Hoxa5promoter

    DNA

    CreRecombinase-ERT2

    Tamoxifenbinds

    Cre-ERT2receptor

    Tamoxifen

    STOPCassette

    LoxP LoxP

    TomatoCDS

    TomatoCDSConstitutivepromoter

    DNA

    DNA

    Figure1:TheInducibleCre-ERT2model

    Cytoplasm

  • Cre-ERT2allowstemporalfatemappingofHoxa5-expressingcells

    EmbryonicStages(days) e7.0e8.5 e9.5e10.5e11.5e12.5e13.5e14.5e15.5

    Hoxa5expressionbegins

    TamoxifenInjection

    Hoxa5-expressingcellslabelledwithCre-ERT2

    HarvestHoxa5 EndogenousExpression

  • DetermineoptimaldoseofTamoxifenforCreinductioninmice

    IdentifyHoxa5descendantsat3

    differenttimepoints

    Methods:Timeline

  • Experiment1: Generate embryosinwhichtheHoxa5 -expressingcellsandtheirdescendantsarelabelledwithTdtomato

    InjectTamoxifenatspecificdose

    (9,12or15mg/40mg)

    Harvestandembedembryos

    inOCT

    Sectionwithcryostat

    Immuno-histochemistry

    MicroscopyandPhotography

    Ate8.5

    Ate14.5

    ChooseTamoxifendosewith AdequateTdtomatolabeling

    Embryossurvive

  • Generating embryosinwhichtheHoxa5-expressingcellsandtheirdescendantsarelabelledwithTdtomato

    XHoxa-5Cre/+Male Tdtomato /Tdtomato Female

    Hoxa-5Cre/Tdtomato embryo

  • Generate embryosinwhichtheHoxa5 -expressingcellsandtheirdescendantsarelabelledwithTdtomato

    InjectTamoxifenatspecificdose

    (9,12or15mg/40mg)

    Harvestandembedembryos

    inOCT

    Sectionwithcryostat

    Immuno-histochemistry

    MicroscopyandPhotography

    Ate8.5

    Ate14.5

  • Tamoxifenwasintraperitoneallyinjectedatdifferent trialdoses(9,12or15mg/40mgofbody weight)ate8.5

    Ate14.5,embryoswereharvestedandembeddedinOCT

    Embryosweresectionedusingacryostat

  • Generate embryosinwhichtheHoxa5 -expressingcellsandtheirdescendantsarelabelledwithTdtomato

    InjectTamoxifenatspecificdose

    (9,12or15mg/40mg)

    Harvestandembedembryos

    inOCT

    Sectionwithcryostat

    Immuno-histochemistry

    MicroscopyandPhotography

    Ate8.5

    Ate14.5

  • Immunohistochemistryformuscleactin

    MicroscopywasperformedtoimagetheTdtomato inHoxa5

    descendants

    Determinewhichdoseproducedoptimalflorescence

  • Immunohistochemistry,MicroscopyandPhotography

    Sectionofe9.5injectedembryowithoptimaltamoxifendoseof9mg/40mgofbodyweight

  • 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

  • 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

  • Results:LabelingHoxa5 expressingcellsate9.5-10.5

  • MuscleActinA5descendants

    MuscleActinA5descendants

    MuscleActinA5descendants

    MuscleActinA5descendants

    MuscleActin

    MuscleActinA5descendants

    MuscleActinA5descendants

    e9.5 e9.5e9.5

    e12.5 e12.5 e13.5 e13.5

  • e9.5 e12.5 e13.5

    MuscleActinA5descendants

    MuscleActinA5descendants

    MuscleActinA5descendants

  • e9.5 e12.5 e13.5

    MuscleActinA5descendants

    MuscleActinA5descendants

    MuscleActinA5descendants

  • Summary:Hoxa5-expressingcellsshowtissue-typerestriction

    TransgenicCre lineandtheCre-ERT2linerevealedtissue-specificrestrictionofcellsthat expressHoxa5

    MyCre-ERT2resultssuggestthatHoxa5expressingcellsgiverisetochondrocytesinatime-dependentmanner

    Dermomyotome Dermis Muscle MuscleConnectiveTissue

    Adipocytes

    Sclerotome Chondrocytes Perichondrium Ligaments Tendons

    = Hoxa5 descendantscontributetothesetissues

    NeuralTube Neurons

  • TissueTypes e9.5 e12.5 e13.5Chondrocytes 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

  • TissueTypes e9.5 e12.5 e13.5Chondrocytes 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.

  • Conclusion

    Hoxa5 patternsregionalidentitybyactingon

    asubsetoftissuetypes

    Hoxa5-expressingcellscontributeto

    chondrocytesinatemporal-specificmanner

  • FutureDirections

    Refinetimepointinvestigationsbyfatemappingateachgestationdaybetweene9.5ande12.5

    IdentifytranscriptionaltargetsofHoxa5inthetissuesandtimepointsthatwehavecharacterizedashavingHoxa5descendants.

  • Implications StudyingtheroleofHoxa5 iscrucialtounderstanding

    Complexities behindembryologicaldevelopment

    Howearlymutationscouldleadtophysicalabnormalities

    andhealthdefects

    Withthisinformation,future doctorscouldpotentially

    treatorpreventphysicalabnormalitiesusinggene

    therapy

  • NationalScienceFoundation HowardHughesSciencePipelineProject MyLabmentor,ProfessorMansfield Mythesismentor,ProfessorRomeo FellowN&Bmajors PastandPresentStudentsoftheMansfieldLab Biology&NeuroscienceDepts. FriendsandFamily!J