Culture conditions affect the cholinergic development of an isolated subpopulation of chick mesencephalic neural crest cells☆

摘要

Althoughneuralcrestcellsareknowntobeveryresponsivetoenvironmentalcuesduringtheirdevelopment,recentevidenceindicatesthatatleastsomesubpopulationsmaybecommittedtoaspecificdifferentiationprogrampriortomigration.Becausetheneuralcrestiscomposedofaheterogeneousmixtureofcellsthatcontributestomanyvertebratecelllineages,assessingthepropertiesofspecificsubpopulationsandtheeffectoftheenvironmentontheirdevelopmenthasbeendifficult.Toaddressthisproblem,wehaveisolatedapuresubpopulationofchickmesencephalicneuralcrestcellsbyfluorescenceno-flowcytometryafterlabelingthemwithmonoclonalantibodies(Mabs)toa75-kDacellsurfaceantigenthatisassociatedwithhighaffinitycholineuptake.WhenculturesofchickmesencephalicneuralcrestcellsarelabeledwiththeseMabsandafluorescentsecondstepantibody,a??5%ofthecellsareantigen-positive(A+).Aftersorting,100%oftheresultingculturedmesencephalicneuralcrestcellsareA+.TheMabsweusedalsolabelalloftheneuronsoftheembryonicchickandquailciliaryganglioninvivoandinvitro.Wehavecomparedtheeffectofvariouscellculturemediaontheisolatedneuralcrestsubpopulationandtheheterogeneouschickmesencephalicneuralcrestfromwhichitwasderived.A+cellswerepassagedandgrowninavarietyofmedia,eachofwhichdifferentlyaffecteditscharacteristicsanddevelopment.A+cellsproliferatedinthepresenceof15%fetalbovineserum(FBS)andhighconcentrations(10a€“15%)ofchickembryoextract,butdidnotdifferentiate,althoughtheyretainedbasallevelsofcholineacetyltransferase(ChAT)activity.However,inchickserumandhigh(25mMasopposedto7mM)K+,andheart-,iris-,orlung-conditionedmedium,allofwhichareknowntopromotesurvivaland/orcholinergicdevelopmentofciliaryganglionneurons,thecellsceasedtoproliferateandallofthecellsintheculturebecamea€?neuron-likea€?within10days.Noneuron-likecellswerefoundinliver-,notocord-,orneuraltube-conditionedmediaifFBSwasused.WhenA+cellswereeliminatedeitherbycomplement-mediatedcytotoxicityorbylaser-ablatingA+cellsduringno-flowcytometry,allChATactivitywasalsoeliminated,andnoneuron-likecellsorChATactivitywasfoundinculturesduringasubsequent3-weekcultureperiod.Weconcludethatcultureconditionsthatnormallypromoteciliaryganglionneuronsurvivalandcholinergicdevelopmentalsopromotedifferentiationanddevelopmentofcholinergicneuron-likecellsfromtheisolatedA+neuralcrestsubpopulation.TheseresultsareconsistentwiththehypothesisthattheA+subpopulationcontributestotheformationofciliaryganglionneuronsintheembryo.