Visualizing Normal and Defective Bone Development in Zebrafish Embryos Using the Fluorescent Chromophore Calcein

Developmental Biology

Yonathan Zohar¹  Greg Kindschi¹  Shao Jun Du¹  Victor Frenkel¹ 

[1] Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland, 21202

关键词: skeletal structure;    bone;    calcein;    zebrafish;    BMP;   

DOI  :  10.1006/dbio.2001.0390

学科分类：生物科学（综合）

来源: Academic Press

【 摘 要 】

Zebrafishhaverecentlybecomeamodelofchoiceamongdevelopmentalbiologists.Thisuniquemodelenablesbothmodernmolecularandgeneticstudiestobecarriedouttoidentifygenesinvolvedinawidevarietyofdevelopmentalprocesses.Thesuccessofthegeneticapproachdependslargelyontheapplicationofaneasyandeffectivescreeningmethodtoidentifyinterestingmutants.Inordertodevelopamethodforvisualizingskeletalstructuresinzebrafishembryosthatwouldbesuitableforscreeningskeletalmutants,weinvestigatedtheuseofthefluorescentchromophorecalcein,whichbindsspecificallytocalcifiedskeletalstructures.Byusingthismethod,wefollowedthedevelopmentoftheskeletalstructuresinzebrafishembryosfromday1today21postfertilization,andanalyzedtheeffectofbonemorphogeneticprotein-2(BMP2)onaxialskeletondevelopment.Wefoundthedevelopmentofthecalcifiedskeletalstructuretoappearinaprogressivefashionfromheadtotail.Calcifiedstructuresinthehead(i.e.,thejaw)developedfirst,whichwerethenfollowedbytheaxialskeletoninthetrunk.Interestingtonotewasthatthereappearedtobetwodomainsinthecalcificationofvertebraewithintheaxialskeleton.Thefirstthreevertebraewereinthefirstdomain;therestbeingintheseconddomain.ComparedwithAlcianbluestaining,wefoundthatcalceinstainingindeedlabelscalcifiedskeletalstructures,and,moreover,itisamoresensitiveandinclusivemethodforvisualizingskeletalstructures.Todeterminewhethercalceinstainingcouldalsobeusedtodetectabnormalbonedevelopment,weectopicallyexpressedBMP2inzebrafishnotochordcells.WedemonstratedthatectopicexpressionofBMP2innotochordcellsinhibitedthedevelopmentoftheaxialskeleton.Together,theseresultsclearlydemonstratedthesensitivityofcalceinstainingforvisualizingbonestructuresindevelopingzebrafishembryosanditseffectivenessforscreeningformutantsthathavebonestructuredefects.

【 授权许可】

Unknown