Changes in extracellular matrix gene and protein expression in developing mouse submandibular glands PDF
Preview Changes in extracellular matrix gene and protein expression in developing mouse submandibular glands
CHANGESINEXTRACELLULARMATRIXGENE ANDPROTEINEXPRESSIONIN DEVELOPINGMOUSESUBMANDIBULAR GLANDS BY SHAWNP.MACAULEY ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 1996 tomywifeBeth forherlimitlesssacrificesandlove ...theveryorder,changes,and movementsintheuniverse,thevery beautyofforminallthatisvisible, proclaim,howeversilently,boththat theworldwascreatedandalsothatits CreatorcouldbenoneotherthanGod whosegreatnessandbeautyareboth ineffableandinvisible. St.Augustine,TheCityofGod ACKNOWLEDGMENTS IoffermysinceregratitudetoDrs.MichaelHumphreys-BeherandGregorySchultz, whohavehelped moldand shape myunderstanding ofsciencethroughtheircomplete support, patience, and guidance during these graduate studies. My sincere thanks are extendedtoDrs.LindaBrinkley,N.IngvarMagnussonandWilliamBuhi,membersofmy committee,fortheirhelpfulsuggestionsoverthepasttwoyears. Averyspecialthankyou goestoDr.RichardBoyce,DepartmentofBiochemistryandMolecularBiology,whohelped smooth the bumps along the way and helped keep everything in perspective. Also instrumentalinmyfinishingthisdegreeprogramisDr.ArnoldS.Bleiweis,Chairmanof OralBiology,whoalongwiththedepartmentfaculty,allowedmetheopportunitytojointhe department. IwouldalsoliketothankDr.GreggOxford,Dr.KarnumPurushotham,Mr.Micah Kerr,Ms.ElizabethBowen,Mr.ChrisRobinson,Mr.JasonBrayer,Ms.Kim-HoaNyugen, Ms.RandiBroverman,Dr.HideoYamamoto,Dr.ZengTao,Dr.Pao-LiWang,Dr.Yanfei Hu,andDr.RongfaBu,allpastandpresentmembersofthelaboratory,formakingscience equallyfunandentertaining. Alongwiththelabpersonnel,specialthanksgotofellow graduatestudentsMs.HeatherAllison,Ms.LoriWojciechowski,Dr.AdrianaDaSilviera, in Ms. Lina Bueno, and Mr. James Kohler who have helped make the graduate student experienceveryenjoyable. SpecialthanksgotomembersoftheInstituteforWoundResearch~Dr.Nasser Chegini,forassistanceinimmunohistochemistryandinsituhybridization,Dr.Roy Tarnuzzer,forassistanceinsynthesizingpMATRIX,andespeciallytoMrs.LandisYoung whohasassistedmeinimmeasurableways. Allofthesecontributionswereinvaluabletothe project. Finally,Iwishtothankaboveall,myLordandSaviorJesusChrist,myparents, KarenandCurtisWinters,myin-laws,BarbaraandJohnMartin,mycherishedwifeBeth, andmydaughtersErinandEmily,fortheirunconditionalloveandenduringstrength. IV TABLEOFCONTENTS page ACKNOWLEDGMENTS Hi LISTOFFIGURES vii ABSTRACT ix CHAPTERS INTRODUCTION 1 1 TheExtracellularMatrix 1 SalivaryGlandDevelopment 16 SalivaryGlandMorphogenesis 18 RoleofEpithelial-MesenchymalInteractionsinSalivary GlandMorphogenesis 18 ContributionoftheExtracellularMatrixtoSalivary GlandMorphogenesis 23 ContributionofGlycosaminoglycanstoSalivary GlandMorphogenesis 30 SalivaryGlandCytodifferentiation 33 RoleofEpithelial-MesenchymalInteractionsinSalivary GlandCytodifferentiation 34 ContributionoftheExtracellularMatrixinSalivary GlandCytodifferentiation 35 ContributionofGlycosaminoglycansinSalivaryGland Cytodifferentiation 36 StatementoftheProblem 38 GeneQuantitation 39 RT-PCR 40 CompetitiveRT-PCR 43 2 MATERIALSAND METHODS 46 SyntheticSupertemplatepMATRIXConstruction 46 SelectionofOligonucleotideSequences 46 SynthesisofMultiprimerTemplate 49 EngineeringandInsertionofaPoly(A)Tail 56 IsolationofPoly(A)+RNA 58 IsolationofTotalCellularRNA 59 QuantitativeRT-PCR 60 Separationofproductsandphotographingofgels 63 Scanningimageandfinalanalysis 63 Statisticalanalysis 63 Immunohistochemistry 64 3 RESULTS 67 SynthesisofpMATRIX 67 QuantitationofECMGeneExpressionUsingpMATRIX 70 ECMProteinLocalizationbyImmunohistochemistry indevelopingmousesubmandibularglands 76 4 DISCUSSION 81 5 OTHERSTUDIES 95 APPENDICES A: ListofOligonucleotidesSynthesizedforpMATRIX 101 B: RestrictionMapofpMATRIXMultiprimerInsert 102 REFERENCES 104 BIOGRAPHICALSKETCH 121 VI LISTOFFIGURES Figure Page 1-1 Structureoffibrillarcollagens 3 1-2 StructureofcollagentypeIV 6 1-3 Structureoffibronectin 8 1-4 Structureoflaminin 10 1-5 Structureofelastin 12 1-6 Crosslinkingreactioncatalyzedbylysyloxidase 13 1-7 Structureofglycosaminoglycans 15 1-8 Invitrodevelopmentofafetalmousesubmandibulargland 20 1-9 Photographofafreshlydissectedday13fetalmouse submandibulargland 21 1-10 Effectsofcollagenaseandcollagenaseinhibitors onsubmandibularglanddevelopment 26 1-11 SchematicdiagramoftheRT-PCRmethod 42 2-1 Alignmentofoligonucleotidestocreatemultiprimerinsert 48 2-2 SchemefortheconstructionofpMATRIX 51 2-3 Nucleotidesequenceof501bpmultiprimerinsert 53 VII 2-4 Cloningofapoly(A)tailtothemultiprimerinsert 57 2-5 GeneralschemeforquantitativeRT-PCRexperiments 62 2-6 Immunohistochemistryprotocol 66 3-1 RepresentativecompositedataforQ-RT-PCR 69 3-2 Ethidiumbromidestainedagarosegelsshowing Q-RT-PCRdataforlamininB2and fibronectinatday18 71 3-3 Graphicalanalysisofdata 72 3-4 CopiesofmRNApercellforeachECMgene measuredoverthedevelopmentofmouse submandibularglands 74 3-5 ProteinlocalizationforECMmolecules 78 4-1 SchematicrepresentationoftheexpressionofECMproteins indevelopingmousesubmandibularglands 87 4-2 Listofotherpotentialsupertemplates 94 Vlll AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy CHANGESINEXTRACELLULARMATRIXGENE ANDPROTEINEXPRESSIONIN DEVELOPINGMOUSESUBMANDIBULAR GLANDS By ShawnP.Macauley May1996 Chair:MichaelG.Humphreys-Beher,Ph.D. MajorDepartment: OralBiology Submandibularglandfetaldevelopmentinvolvestheorchestratedexpressionof extracellularmatrixmolecules(ECM)whichdirectthemorphogenesis,differentiation, andbuddingoftheepithelium. Earlymorphogenesisofmousesubmandibularglands beginsonlateday11ofdevelopmentwhentheepitheliumbeginstobudfromthe surroundingmandibularmesenchyme. Submandibularglandshavetwoburstsof development,oneinuteroandasecondpostnatallyoccurringinthesuckling/weanling transition.FromthesmallamountsoftotalRNAcollectedfromfetalBALB/c submandibularglands,geneexpressionwasmeasuredusingquantitativecompetitive RT-PCR.ToinvestigatethechangesinECMgeneexpressionduringfetaldevelopment, amultiprimerplasmid(pMATRIX)wasconstructedwhichcontainstheprimer IX sequencesforcollagensa2(I),al(III),al(IV),fibronectin,lamininsBlandB2,elastin, andlysyloxidase. Theprimersequenceswereidenticalforhuman,rat,andmouse. cRNAwassynthesizedfromthesupertemplateandaddedinvaryingknownamountsto aseriesofreversetranscriptionreactionseachcontaining1.0ugoftotalsubmandibular glandRNA. BycomparingthePCRamplificationproductsofboththecellularmRNA andthesynthetictemplate,itwaspossibletomeasurethedirectexpressionofeachgene. Thenumberofcollagencc2(I),collagenal(III),andlysyloxidasemRNAmoleculesper cellpeakedonday16ofdevelopment. TheexpressionofelastinmRNAwasdetectable onlyonday16. FibronectinandlamininB2weremoreconstitutiveintheirexpression butagainhadtheirhighestcopynumberpercellonday16. AsecondburstofECM geneexpressionoccurredintheneonate,withthelowestECMgeneexpressionseenin thematureadultgland. Inaddition,immunohistochemistrywasperformedtolocalize theproteinforeachoftheECMmoleculesexamined. Withtheconstructionofthe pMATRIXsupertemplateandtheadventofcompetitiveRT-PCRtechnologyithasbeen possibletomeasuresmallchangesinECMgeneexpressionduringsalivarygland development. ThisplasmidcanbeutilizedtoexaminetheECMgeneexpressionofany human,mouse,orrattissuepreviouslythoughttobetoosmallorlimitedfortraditional techniques.
The list of books you might like
