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Methods in Molecular Biology 2403 Sebastian Dworkin Editor Craniofacial Development Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Craniofacial Development Methods and Protocols Edited by Sebastian Dworkin La Trobe University, Bundoora, VIC, Australia Editor SebastianDworkin LaTrobeUniversity Bundoora,VIC,Australia ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-1846-2 ISBN978-1-0716-1847-9 (eBook) https://doi.org/10.1007/978-1-0716-1847-9 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2022 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Mostmulticellularorganismscompriseadefined“head”region.Theformationanddevel- opment of this craniofacial region, comprising the face, jaws, nerves, blood vessels, and musculature (and in vertebrates also the skull and facial bones), requires an essential, integrated network of precisely regulated developmental cues and signals. The study of craniofacialbiologythereforenecessitatesamultitudeofcomplementaryapproachestofully understandhow allthemolecular and cellularcomponents acttogether to ultimatelyform the head and face. The use of animal models as disparate as nematodes (Caenorhabditis elegans), zebrafish (Danio rerio), and mice (Mus musculus), as well as advanced imaging of thedevelopingskullinhumanpatients,hasprovidedanincrediblewealthofknowledgeto allow us to understand how the human face develops and, importantly, also begin to understand what happens when things go wrong—resulting in developmental anomalies, termedcraniofacialdefects. This book will explore and characterize protocols in craniofacial biology that cover topics including anterior-posterior polarity in facial establishment (C. elegans), molecular signalsthatunderpincraniofacialprimordiaestablishmentanddevelopment(zebrafish),and themorecomplexmechanismsofneuraltubeclosure,palataldevelopmentandfusion,and mammaliancraniofacial skeletonformation (mouse). This bookwill also includea number ofchapterscoveringtechniquesemployedintheclinic,includingcomputationalanalysesof craniosynostosis, bone microarchitecture, and X-ray micro-computed tomography (micro- CT).Furthermore,thisbookincludeschaptersoninvestigatingcellulardynamicsofcranio- facial cells, including osteoblasts, cells of the neural crest and salivary glands, craniofacial primordia explant cultures, methods for bone regeneration using cryogel scaffolds, and techniquesforinvestigatingtherelationshipbetweenepithelialandmesenchymalcellpopu- lations. Several chapters cover histological procedures and useful staining techniques in zebrafish and mouse models, and genomic editing is presented in two chapters outlining the identification of genomic elements that drive gene expression and gene deletion approaches via CRISPR/Cas9 in zebrafish and mouse. Lastly, a chapter is presented on bioinformatic methodology currently employed to integrate genetic and molecular infor- mationthroughsingle-cellRNAsequencing(scRNA-SEQ). Ultimately,thisbookisintendedtoserveasa“go-to”guideforstudentsinthefieldof craniofacial development, while also acting as an essential guide to bridge the often-large divide between basic science researchers and doctors and surgeons in clinical practice. Ultimately,we envisionthisbookmay alsobeofuseto more senior researcherslookingto expandtheirexistingresearchprogramstoencompassnoveltechniques. Bundoora,VIC,Australia SebastianDworkin v Contents Preface ..................................................................... v Contributors................................................................. ix 1 UsingCaenorhabditiselegansasaModelforMechanisticInsights ofCraniofacialDevelopment ............................................. 1 MichaelGrussandAnnK.Corsi 2 WholemountIn-SituHybridization(WISH)inZebrafishEmbryos toAnalyzeCraniofacialDevelopment...................................... 19 NishanthiMathiyalaganandSebastianDworkin 3 ElucidationofGeneExpressionPatternsintheCraniofacialTissues ofMouseEmbryosbyWholemountInSituHybridization................... 33 JoshuaB.Studdert,HeidiBildsoe,V.PragathiMasamsetti, andPatrickP.L.Tam 4 VisualizationoftheCartilageandBoneElementsintheCraniofacial StructuresbyAlcianBlueandAlizarinRedStaining......................... 43 JoshuaB.Studdert,HeidiBildsoe,V.PragathiMasamsetti, andPatrickP.L.Tam 5 MethodologyforFree-FloatingOrganCultureofMid-gestation MaxillaryPrimordialTissue .............................................. 51 M.E.deVriesandSebastianDworkin 6 MethodsofPalateCultureinLaterPalatogenesis:Elevation, HorizontalOutgrowth,andFusion ....................................... 63 JackD.MorganandJeremyB.A.Green 7 SingleCellRNA-Seq:CellIsolationandDataAnalysis ...................... 81 ValYianniandPaulT.Sharpe 8 GeneratingZebrafishRNA-LessMutantAllelesbyDeleting GenePromoterswithCRISPR/Cas9...................................... 91 PriyankaKumari,MorganSturgeon,GregoryBonde, andRobertA.Cornell 9 CraniofacialPhenomics:Three-DimensionalAssessment oftheSizeandShapeofCranialandDentofacialStructures.................. 107 MustafaMian,JennyTan,RobinYong,RuthWilliams, AgathaLabrinidis,PeterJ.Anderson,andSarbinRanjitkar 10 Micro-CT–BasedBoneMicroarchitectureAnalysisoftheMurineSkull........ 129 JennyTan,AgathaLabrinidis,RuthWilliams,MustafaMian, PeterJ.Anderson,andSarbinRanjitkar vii viii Contents 11 CharacterizationofMammalianInVivoEnhancersUsingMouse TransgenesisandCRISPRGenomeEditing................................ 147 MarcoOsterwalder,StellaTran,RianaD.Hunter,EmanM.Meky, KiannavonMaydell,AnneN.Harrington,JanethGodoy, CatherineS.Novak,IngridPlajzer-Frick,YiwenZhu, JenniferA.Akiyama,VeenaAfzal,EvgenyZ.Kvon, LenA.Pennacchio,DianeE.Dickel,andAxelVisel 12 HistologicalTechniquesforSectioningBonesoftheVertebrateCraniofacial Skeleton ............................................................... 187 DanielaM.Roth,LakshmiPuttagunta,andDanielGraf 13 EngineeringEpithelial–MesenchymalMicrotissuestoStudyCell–Cell InteractionsinDevelopment............................................. 201 JacobI.Reynolds,RossA.Vitek,PeterG.Geiger,andBrianP.Johnson 14 ExVivoCultureofHumanCranialSutureCells............................ 215 PeterJ.Anderson 15 ScaffoldsforUseinCraniofacialBoneRegeneration ........................ 223 KatherineR.Hixon,ChristopherT.Eberlin,MeghanaPendyala, AngelaAlarcondelaLastra,andScottA.Sell 16 MandibleExplantAssayfor theAnalysisofMeckel’sCartilage Development........................................................... 235 SophieWiszniakandQuentenSchwarz 17 High-ResolutionHistologyforCraniofacialStudiesonZebrafish andOtherTeleostModels ............................................... 249 A.Huysseune,M.Soenens,J.-Y.Sire,andP.E.Witten 18 LiveImagingoftheDynamicsofMammalianNeuralCrestCell Migration.............................................................. 263 EmmaL.Moore andPaulA.Trainor 19 SalivaryGlandDevelopmentinCulture ................................... 277 MarciaGaete,TathyaneH.N.Teshima,LemoniaChatzeli, andAbigailS.Tucker 20 AntenatalUltrasoundImagingforAnalysisofHumanCraniosynostosis....... 295 SarahConstantineandPiotrNiznik Index ...................................................................... 311 Contributors VEENA AFZAL • EnvironmentalGenomicsandSystemsBiologyDivision,LawrenceBerkeley NationalLaboratory,Berkeley,CA,USA JENNIFERA.AKIYAMA • EnvironmentalGenomicsandSystemsBiologyDivision,Lawrence BerkeleyNationalLaboratory,Berkeley,CA,USA ANGELAALARCON DELALASTRA • DepartmentofImmunology,WashingtonUniversity,St. Louis,MO,USA PETERJ.ANDERSON • AdelaideDentalSchool,TheUniversityofAdelaide,Adelaide,SA, Australia;AustralianCraniofacialUnit,Women’sandChildren’sHospital,North Adelaide,SA,Australia;SouthAustralianHealthandMedicalResearchInstitute (SAHMRI),Adelaide,SA,Australia HEIDIBILDSOE • CentreforReproductiveHealth,HudsonInstituteofMedicalResearch, Clayton,VIC,Australia GREGORYBONDE • DepartmentofAnatomyandCellBiology,UniversityofIowa,IowaCity, IA,USA LEMONIACHATZELI • WellcomeTrust/CancerResearchUKGurdonInstitute,Universityof Cambridge,Cambridge,UK SARAH CONSTANTINE • DepartmentofMedicalImaging,Women’sandChildren’sHospital, NorthAdelaide,SA,Australia ROBERTA.CORNELL • DepartmentofAnatomyandCellBiology,UniversityofIowa,Iowa City,IA,USA ANNK.CORSI • DepartmentofBiology,TheCatholicUniversityofAmerica,Washington, DC,USA M.E.DEVRIES • DepartmentofMedicine,MonashUniversityCentralClinicalSchool, Prahran,VIC,Australia;DepartmentofPhysiology,AnatomyandMicrobiology,LaTrobe University,Melbourne,VIC,Australia DIANEE.DICKEL • EnvironmentalGenomicsandSystemsBiologyDivision,Lawrence BerkeleyNationalLaboratory,Berkeley,CA,USA SEBASTIANDWORKIN • DepartmentofPhysiology,AnatomyandMicrobiology,LaTrobe University,Bundoora,VIC,Australia CHRISTOPHERT.EBERLIN • UniversityofIllinoisCollegeofMedicine,Peoria,IL,USA MARCIAGAETE • DepartmentofAnatomy,FacultyofMedicine,PontificiaUniversidad Cat(cid:1)olicadeChile,Santiago,Chile PETERG.GEIGER • DepartmentofBiomedicalEngineering,UniversityofWisconsin, Madison,WI,USA JANETHGODOY • EnvironmentalGenomicsandSystemsBiologyDivision,LawrenceBerkeley NationalLaboratory,Berkeley,CA,USA DANIELGRAF • SchoolofDentistry,UniversityofAlberta,Edmonton,AB,Canada; DepartmentofMedicalGenetics,UniversityofAlberta,Edmonton,AB,Canada JEREMYB.A.GREEN • CentreforCraniofacialandRegenerativeBiology,King’sCollege London,London,UK MICHAELGRUSS • DepartmentofBiology,TheCatholicUniversityofAmerica,Washington, DC,USA ix x Contributors ANNE N.HARRINGTON • EnvironmentalGenomicsandSystemsBiologyDivision,Lawrence BerkeleyNationalLaboratory,Berkeley,CA,USA KATHERINER.HIXON • DepartmentofOrthopaedicSurgery,WashingtonUniversity,St. Louis,MO,USA;ThayerSchoolofEngineering,DartmouthCollege,Hanover,NH,USA RIANA D.HUNTER • EnvironmentalGenomicsandSystemsBiologyDivision,Lawrence BerkeleyNationalLaboratory,Berkeley,CA,USA A.HUYSSEUNE • BiologyDepartment,ResearchGroupEvolutionaryDevelopmentalBiology, GhentUniversity,Ghent,Belgium BRIAN P.JOHNSON • DepartmentofBiomedicalEngineering,InstituteforQuantitative HealthScienceandEngineering,MichiganStateUniversity,EastLansing,MI,USA; DepartmentofPharmacology&Toxicology,MichiganStateUniversity,EastLansing, MI,USA PRIYANKAKUMARI • DepartmentofAnatomyandCellBiology,UniversityofIowa,IowaCity, IA,USA EVGENYZ.KVON • EnvironmentalGenomicsandSystemsBiologyDivision,Lawrence BerkeleyNationalLaboratory,Berkeley,CA,USA;DepartmentofDevelopmentalandCell Biology,UniversityofCalifornia,Irvine,Irvine,CA,USA AGATHA LABRINIDIS • AdelaideMicroscopy,TheUniversityofAdelaide,Adelaide,SA, Australia V.PRAGATHIMASAMSETTI • EmbryologyUnit,Children’sMedicalResearchInstitute, Westmead,NSW,Australia NISHANTHIMATHIYALAGAN • DepartmentofPhysiology,AnatomyandMicrobiology,LaTrobe University,Bundoora,VIC,Australia EMANM.MEKY • EnvironmentalGenomicsandSystemsBiologyDivision,LawrenceBerkeley NationalLaboratory,Berkeley,CA,USA MUSTAFAMIAN • AdelaideDentalSchool,TheUniversityofAdelaide,Adelaide, SA,Australia EMMA L.MOORE • StowersInstituteforMedicalResearch,KansasCity,MO,USA JACKD.MORGAN • CentreforCraniofacialandRegenerativeBiology,King’sCollege London,London,UK PIOTRNIZNIK • DepartmentofMedicalImaging,Women’sandChildren’sHospital,North Adelaide,SA,Australia CATHERINES.NOVAK • EnvironmentalGenomicsandSystemsBiologyDivision,Lawrence BerkeleyNationalLaboratory,Berkeley,CA,USA MARCOOSTERWALDER • EnvironmentalGenomicsandSystemsBiologyDivision,Lawrence BerkeleyNationalLaboratory,Berkeley,CA,USA;DepartmentforBioMedicalResearch (DBMR),UniversityofBern,Bern,Switzerland MEGHANA PENDYALA • DepartmentofBiomedicalEngineering,RensselaerPolytechnic Institute,Troy,NY,USA LENA.PENNACCHIO • EnvironmentalGenomicsandSystemsBiologyDivision,Lawrence BerkeleyNationalLaboratory,Berkeley,CA,USA;USDepartmentofEnergyJointGenome Institute,LawrenceBerkeleyNationalLaboratory,Berkeley,CA,USA;Comparative BiochemistryProgram,UniversityofCalifornia,Berkeley,CA,USA INGRID PLAJZER-FRICK • EnvironmentalGenomicsandSystemsBiologyDivision,Lawrence BerkeleyNationalLaboratory,Berkeley,CA,USA