Dima Chaar Senior Thesis presentation
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Transcript of Dima Chaar Senior Thesis presentation
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
Dermomyotomeà• Dermis• Muscle• MuscleConnectiveTissue
• Adipocytes
Sclerotomeà• Chondrocytes(cartilage)
• Perichondrium• Ligaments• Tendons
NeuralTubeà• 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 Hoxa5’s role in somite differentiation
•Weknow:
• WhereHoxa5 isexpressed
• WhatrolesHoxa5 performsinvertebraldevelopmentfromstudiesof
loss-of-functionmutations
•Wedon’tknow:
• HowHoxa5 playsitscrucialroleinvertebraldevelopment
• WhichtissuesarederivedfromHoxa5-expressingcellsandwhenisHoxa5
expressionnecessary?
• 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
Dermomyotomeà• Dermis• Muscle• MuscleConnectiveTissue
• Adipocytes
Sclerotomeà• Chondrocytes• Perichondrium• Ligaments• Tendons
✓
✓
✓
✓
✓= Hoxa5 descendantscontributetothesetissues
NeuralTubeà• Neurons✓
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
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.
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