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