AnIntroductiontoModelOrganismsofDevelopmentGas:2000litersofmethanegasreleased/day!

Whatmakesagoodmodelorganism?Gas:2000litersofmethanegasreleased/day!

Size:6tons250kgfoodeatenevery100kgofelephantdung/dayGestation:23monthsFemalesgivebirthtosingleoffspringeveryfiveyearsSexualmaturityatage12Whatmakesagoodmodelorganism?Size:1mminlength

LiveonadietofbacteriaGestation:500,000offspringin1weekfromsingleorganismSexualmaturityin3daysGenome:

Sequenced!DrosophilamelanogasterCaenorhabditiselegansArthropodsNematodesXenopuslaevisAmphibiansMammalsMusmusculusHomosapiensCaenorhabditiselegansNematodeWormNematodesaccountforanestimatedfourofeveryfiveanimalsintheworld!Smooth-skinned,unsegmentedwormsfirstusedasamodelorganismbySydneyBrennerin1965C.elegansisdiploidandhasfivepairsofautosomalchromosomes(namedI,II,III,IVandV)andapairofsexchromosomes(X).Mostadultsarehermaphrodite(XX)but.05%oflabpopulationsaremale(XO)Lifespanis2to3weeksWormsareusuallykeptonpetriplatesandfedE.coliAbout10,000wormsfitonasingleplate959Somaticcells-allvisiblewithmicroscope-300neurons-81musclecells-131cellsundergoprogrammedcelldeathDevelopmentalfateofeverycellisknowninC.elegans8to17roundsofdivisionarerequiredforcelldifferentiationdependingontissuetypeandcellfunction

C.elegansgenomecontains19,000genesandisfullysequenced70%ofhumangeneshavewormhomologues

C.eleganswillbethefirstandpossiblyonlyanimalthatweknoweverythingaboutatacellularandmolecularlevelGenefunctionstudieshavebecomerelativelysimplewiththerecentdiscoveryofRNAiUsingRNAinterferenceforlocalandsystemicgenesilencing

inC.elegansA.C.eleganshermaphroditeexpressingGFPtransgenesinthepharynxandthenucleiofbody-wallmusclecellsB.C.eleganshermaphroditeexpressingGFPtransgenes+GFPdoublestrandedRNAinthepharynxA.B.SmallRNAsasregulategeneexpressionduringdevelopment

Lookforheterochronicdefectsinmutagenesisscreen--cellsbehaveasifinanearlierorlaterdevelopmentalstageRegulatorycascadesunveiledwhichinvolvesmallRNAs(21-22nts)Lin-4andLet-7encodeshortuntranslated

RNAsandfunctionbybindingtocomplementarysequencesinmRNAsofspecificgenescontrollingdevelopmentLin-4expressionallowscellstoprogressfromlarvalstage1to3Let-7expressionallowscellstoprogressfromlatelarvaltoadultstagesAAAAALin-4DevelopmentalmRNANotranslationofmRNAMoleculargeneticoflife-spaninC.elegansC.eleganscanlive2Xlongerundersomeexperimentalconditions:MutationsinDAF-16,AGE-1,DAF-2,orCLK-1;heatshockproteins;feedingbehavior;freeradicalexposureandoxidativestressPathwaysinvolvedappeartobeconnectedGeneralfeaturesoflongerlifespanincludelessreproduction,lessgrowthandmoreDNA/cellularrepairCouldberelatedtocaloricrestrictionobservationsinmammalsDrosophilamelanogasterCommonFruitFlyMoststudiedanimalmodelLifeCycleof2weeks(fertilizationtosexualmaturity)Fourpairsofchromosomes:theX/Ysexchromosomesandtheautosomes2,3,and414,000Genes,sequencedgenomeand2/3ofhumandiseasegeneshaveflyhomologuesLargerepositoriesofmutantfliesavailableConservationofpatterningbetweenfliesandmammalsInsituhybridizationofwholeembryocanrevealpatternsofgeneexpressionduringdevelopmentRNAorDNAprobesandlabeledantibodiesareused.PolyteneChromosomes

PresentinsalivaryglandsoffliesOriginatefromchromosomalduplicationwithnocelldivisionHavepatternsofdarkandlightbandsuniqueforeachchromosomalsectionvisiblewithalightmicroscopeCanbelabeledwithnucleicacidprobesCanbeusedtodeterminebindingsiteoflabeledproteinsChromosomalrearrangmentsanddeletionscanbevisualizedAntennapediamutant:Antennaaretransformedintometathoracic(secondsecondthoracicsegment)legsWildtypeflyStudyingOrganogenesisinDrosophilaImaginaldiscsaregroupsofundifferentiatedcellsinlarvathatgiverisetoadultorgansandstructuresTransplantationandgenemis-expressionstudiesallowcharacterizationoforganformationatcellandmolecularlevelOrgan-specificgeneshavemammalianhomologuesXenopus

LaevisAfricanclawedtoadAdvantagesofusingXenopusasamodel:Vertebratemodelwithfundamentalfeaturesofland-dwellingvertebrates

OocytesarelargeandundergoexternaldevelopmentFemalescanbestimulatedtoovulatewithhormonesDevelopmentisrapid;fertilizationtofullyformedtadpoleinafewdaysLargesizeallowsstudyofmovementofcellswithinXenopusembryos.Cleavageevery30minutes

Gastrulationat10hours1daytoneurulationGermlayersandstructuralcharacteristicsareeasilyobservedManipulationofembryomayinvolvesurgeryormRNAinjectionStudyofXenopusDevelopmentbyEmbryoInjections

mRNAsandcDNAscanbeinjectedtostudyroleofgenesandproteins

AntisensetoknockoutexpressionOver-andmis-expressionofproteinofinterestAlternateproteinforms:dominantnegative,constitutivelyactive,etc.Reporterforms(GFP,etc.)StudymaternalvszygoticcontributionsSignalingmoleculesandchemicalagentscanbeappliedtodetermineaffectsondevelopmentTheEmbryonicSignalingCenter:Spemann’sOrganizer

ClassicexperimentfirstperformedbySpemannandMangoldin1924Grafteddorsallipofanembryoontoasecondembryo

GastrulationinitiatedatbothsitesSecondwholesetofbodystructuresformedCellfatestudiesinXenopus:NogginNogginexpressionpermitscellstobecomebrainandnervoussystemtissueNoNogginexpressionresultsintissuebecomingskin,boneNogginisaninhibitorofBMPswhichpromotebonegrowthUsenucleicacidmicroinjectiontoknockoutorover-expressnogginMusMusculus

BestmodelformammaliandevelopmentLifecycleapproximately9weeks;21daygestationLittersupto20pupsGenomesequencedManyinbredstrainscharacterized(450available)GeneticmanipulationswelldevelopedHouseMouseEmbryoscanbeperturbedinvariouswaysbutgiverisetonormalmouse

AllowsgeneticmanipulationofembryopossibleEarlyembryocanbesplittoyieldtwo“twins”TwomorulascanbecombinedtoformachimeraCellsfromanembryocanbeinjectedintoanotherblastocysttoformachimeraMouseembryosasasourceofembryonicstemcells

Cultureinnercellmass(givesrisetowholeembryo)EScellswilldivideindefinitelywithoutdifferent