Contents lists available atScienceDirect
Seminars
in
Cell
&
Developmental
Biology
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b
Sculpting
the
labyrinth:
Morphogenesis
of
the
developing
inner
ear
Berta
Alsina
a,∗,
Tanya
T.
Whitfield
b,∗aLaboratoryofDevelopmentalBiology,DepartamentdeCiènciesExperimentalsidelaSalut,UniversitatPompeuFabra,ParcdeRecercaBiomèdicade Barcelona,08003Barcelona,Spain
bBatesonCentreandDepartmentofBiomedicalScience,UniversityofSheffield,Sheffield,S102TN,UK
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received31May2016
Receivedinrevisedform26July2016 Accepted25September2016 Availableonlinexxx
Keywords: Innerear Oticplacode Neurogenesis Sensoryhaircell Semicircularcanal Morphogenesis
a
b
s
t
r
a
c
t
Thevertebrateinnerearisaprecisionsensoryorgan,actingasbothamicrophonetoreceivesoundand anaccelerometertodetectgravityandmotion.Itconsistsofaseriesofinterlinked,fluid-filledchambers containingpatchesofsensoryepithelia,eachwithaspecialisedfunction.Theearcontainsmanydifferent differentiatedcelltypeswithdistinctmorphologies,fromtheflask-shapedhaircellsfoundinthickened sensoryepithelium,tothethinsquamouscellsthatcontributetonon-sensorystructures,suchasthe semicircularcanalducts.Nearlyallcelltypesoftheinnerear,includingtheafferentneuronsthatinnervate it,arederivedfromtheoticplacode,aregionofcranialectodermthatdevelopsadjacenttotheembryonic hindbrain.Astheeardevelops,theoticepitheliagrow,fold,fuseandrearrangetoformthecomplex three-dimensionalshapeofthemembranouslabyrinth.Muchofourcurrentunderstandingoftheprocessesof innerearmorphogenesiscomesfromgeneticandpharmacologicalmanipulationsofthedevelopingearin mouse,chickenandzebrafishembryos.Thesetraditionalapproachesarenowbeingsupplementedwith excitingnewtechniques—includingforcemeasurementsandlight-sheetmicroscopy—thatarehelping toelucidatethemechanismsthatgeneratethisintricateorgansystem.
©2016TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).
Contents
1. Introduction...00
2. Segregationoftheoticplacodefromthepre-placodalregion(PPR) ... 00
2.1. Cellmovements...00
2.2. Placodeformation...00
2.2.1. Coalescenceintoaplacode...00
2.2.2. Placodalthickeningandinterkineticnuclearmigration...00
2.2.3. Invaginationandhollowing...00
3. FormationoftheVIIIthganglion:delaminationandmigrationofneuroblasts...00
4. Segregationofsensoryepitheliaandmorphogenesisofsensorychambers...00
5. Morphogenesisofthesemicircularcanals...00
6. Biomechanicsandliveimaging:convergingapproachestounderstandmorphogenesis...00
Acknowledgements ... 00
References...00
1. Introduction
Inanystudyoforganogenesis,itisimportanttogainan appre-ciationnotonlyofthegeneticcontrolofpatterningbutalsoofthe
∗Correspondingauthors.
E-mailaddresses:[email protected](B.Alsina),t.whitfield@sheffield.ac.uk (T.T.Whitfield).
morphogeneticeventsthatgiverisetothethree-dimensionalform ofthematureorgansystem.Understandingthecouplingof sig-nallingpathwaysandtranscriptionfactornetworkactivitytothe cellbehavioursandphysicalforcesthateffectthesemorphogenetic eventsisthusoneofthemajorchallengesinthefield.The devel-opmentofnewtechnologies,particularlyinliveimaging,isnow openingupnewpossibilitiesfortacklingthesechallenges.Forthe inner ear,suchstudieshaveclinicalrelevance: congenital hear-inglosscanalsobeaccompaniedbysomeformofmorphological
http://dx.doi.org/10.1016/j.semcdb.2016.09.015
anomaly,suchasMondinidysplasia(incompletepartitionorcoiling ofthecochlea).Aplasiaofthesemicircularcanalsorwholelabyrinth canalsocauseseveredisruptionofvestibularfunction.
Inthisreview,weconsideraselectionofthemorphological rear-rangementsthattakeplaceastheinner eardevelops.We focus onfourtopics: formationof theotic placode and vesicle; neu-rogenesis and generation ofthe VIIIth ganglion;segregation of sensoryepithelia;andformationofthesemicircularcanalducts. Wehaveomitteddiscussionofanumberofotherimportant pro-cesses,includingformation ofthe endolymphaticduct and sac, establishmentoftheprecisecytoarchitectureofthemammalian organofCorti,theroleofsurroundingtissues(includinghindbrain and periotic mesenchyme),and themorphogenesis of ancillary structures,eachofwhichwouldjustifyaseparatereviewinitsown right.Weendwithaperspectiveonthenewmethodologiesthat arepushingtheboundariesofourunderstandingofhowpatterning iscoupledtomorphogenesisinthedevelopinginnerear.
2. Segregationoftheoticplacodefromthepre-placodal region(PPR)
2.1. Cellmovements
Theoticplacodetogetherwithothercranialplacodes (adenohy-pophyseal,olfactory,lens,trigeminal,epibranchial,oticandlateral line)andtheneuralcrestgiverisetotheelementsofthecranial peripheralnervoussystem.Thecranialplacodesdonot develop directlyasindividualentitiesfromtheectodermbutemergefrom thecommonpre-placodalregion(PPR),ahorseshoe-shaped sub-domainoftheectodermadjacentandlateraltotheneuralplate andneuralcrest[1–5].ThePPRexpressesacombinationof tran-scriptionfactorsoftheSix1/2,Six4/5,Dach,Eya, Dlx,Gata, and Foxifamiliesthatconferitsidentityandcompetenceforspecific placode-inducingsignals[6–14].Thelatter,byactinguponthePPR precursors,drivethesplittingofthePPRandemergenceof individ-ualplacodalfates[15–17].ThesegregationofthePPRintoplacodes isprogressive.Atthelevelofthehindbrainforexample,priortothe appearanceoftheoticplacode,alargePax2/8-expressingdomain encompassestheprecursorsoffuture epibranchialandotic pla-codes (also laterallineprecursors in anamniotes). Thisdomain hasbeencoinedtheotic-epibranchialprecursordomain(OEPD) tohighlighttheclosedevelopmentalrelationshipbetweenthese placodes[9,10,18–20].Theinductiveeventsinvolvedinthe devel-opmentoftheOEPDandoticplacodearereviewedelsewherein thisissue[21];wefocushereonthemorphogeneticmovements leadingtothesegregationofthelargePPRintodiscreteplacodes withinthecranialectoderm.
Fatemappingofpre-placodalprecursorsinchickindicatesthat oticprecursorsareinterspersedwithfutureneuraltissue,neural crestandotherplacodalcellsuntilthefour-somitestage; exten-sivecellmovementshavebeenobservedtoaccompanyplacode development,enablingthesegregationofthedifferentcelltypes [22].Atearlystages(stage5–6inchick),oticprecursorswerefound overalargeterritoryofthePPR,atthelevelofrhombomeres2–7 ofthehindbrain,butwerethenprogressivelyrestrictedtoform theoticplacodeatthelevelofrhombomeres5–6.Convergenceof lateralcellstomedialpositionswasthemostdramaticcell move-ment,accompaniedbysplittingandcellmixingbetweengroupsof cells.Inzebrafish,liveimagingofcellsexpressingGFPdrivenbythe
pax2apromoterwithintheOEPDshowedthatmostGFP-positive
cellsconvergefromanterior,posteriorandlateralpositionstoform theoticplacode,butmoreanteriorandposteriorGFP-positivecells alsocontributetoepibranchialganglia[23].AnalysisofPax2a pro-teinexpression,togetherwithheat-shock-inducedmis-expression andmorpholino-basedgeneknockdown,demonstratedthatcells
withhighlevelsofPax2aproteinhaveatendencytocontribute totheoticplacode,whilelowerlevelsofPax2abiasprecursorsto theepibranchialplacodes[20].ExactlyhowthelevelsofPax2acan influencethesortingand/orconvergenceofpre-placodal precur-sorsneedstobeinvestigatedfurther,butprobablyinvolveschanges incelladhesivity.Interestingly,amorpholino-basedstudysuggests thatdirectedcellmovementsandconvergenceofpre-otic precur-sorstoformthezebrafishoticplacodereliespartlyonthefunction oftheextracellularmatrixreceptorIntegrin-␣5[23].
Similarcellularmovementsleadingtothesegregationof inter-mingledanteriorPPRprecursorsintotheanteriorcranialplacodes (olfactory, lens, adenohypophyseal, trigeminal) have also been described[3,24–26].Theextentofthedirectionalcellmigration dif-fersbetweenspecies;inXenopusandzebrafish,cellmovementsare restrictedtosmallareasandnolarge-scalecellsortingisdetected [17,20].Differencescouldberelatedtothespeciesortotheperiods inwhichmovementshavebeenanalysed,whicharelimitedprior toplacodecoalescence.WhencellsoftheOEPDdisplaysmall-scale movements,theycontributetodistinctoticregionsdependingon theirinitialanteroposteriorlocationintheOEPD,themost ante-riorcellsbeingpreferentiallyallocatedtotheanteriorneurogenic domainandstatoacousticganglion(SAG)[20]andnottothe pos-terior domain of the inner ear. In conclusion, while migratory movementshaverecentlybeenfollowedinrealtime,the under-lyingmoleculesinvolvedinthechemotaxis,sortingandcollective movementsarelittleknown.Moreover,itisstilldebatedwhether PPRcells are already lineage-restrictedbeforetheirsorting out orwhetherrandom movementsfavourtheirpositionalongthe anteroposterioraxis,followedbythereceptionofdistinctsignals thatdirectcellstospecificplacodalfates(seealso[27]).
2.2. Placodeformation
2.2.1. Coalescenceintoaplacode
Howdoplacodesappearasaclusterofcellsafterthesegregation ofPPRcells?Comparedwithotherplacodes,morphogeneticevents ofoticplacodeformationhavereceivedscantattention,butstudies onotherplacodeshintatgenericmechanisms.Whitlockand West-erfieldhaveshownthatbeforethefinalappearanceoftheolfactory placode,olfactoryprecursorsextendoveralongandthinterritory thatprogressivelyconvergestoashorterandwiderdomainalong theanteroposterioraxisandmediolateralaxisrespectively[24].A similareventtakesplaceduringthecoalescenceorconvergence oftheoticplacode.AlvarezandNavascuésfoundlongcytokinetic bridgesduringthisprocess,andhaveproposedalinkbetween cel-lulardisplacementsaftermitosisandplacodeformation[28].Inline withthis,recentimagingofconvergencemovementsduringchick gastrulationidentifiedmitosisasadriverforepithelial rearrange-ments[29].Thishighlightstheneedtore-evaluate,withmodern imagingtechniques,thecontributionofcelldivisionorientation andshapestooticplacodalmorphogenesis.
closeassociationwitheachotherthroughexpressionofhighlevels ofN-cadherin,whiletheneuralcrestcellsexpressthechemokine receptorCxcr4andestablishtransientcell–cellcontactswiththe placodalcells.This“chaseandrun”mechanismisthoughtto con-tributetothefinalpositionoftheplacodeandthecoalescenceof epibranchialprecursorsintoadefinedplacode. Itremains tobe investigatedwhetherasimilarinteractionoccursbetweentheotic placodalprecursorsandthesurroundingneuralcrest,butthelack ofexpressionofSdf1inthezebrafishandchickoticplacode sug-geststhatothermechanismsmightbeinvolvedincoalescenceof theoticplacode[33,34].Eph/ephrinsignalling,whilenottested, isagoodcandidate.Severalmembersofthissignallingpathway areexpressedatoticplacodeandvesiclestages,aswellasin sur-roundingtissues[35].Itremains,thus,tobecharacterisedwhether Eph/ephrinsmediatesortingofoticplacodalcellsand/or segrega-tionfromthesurroundingOEPDcells,ashappensduringhindbrain development[36].
2.2.2. Placodalthickeningandinterkineticnuclearmigration
Afterthecoalescenceofoticprecursors,thechickandmouse oticplacodebecomes visibleand distinctfrom thesurrounding non-placodalectodermasathickenedregion[37].Whilea ‘thicken-ing’describescranialplacodesofamniotes(chickandmouse)well, wherepresumptiveoticcellstransitfromasquamous-cuboidalto acolumnarshape,inzebrafish,theoticplacodeappearstoemerge fromthe unorganised ectodermal cells beneath the enveloping layer(EVL)asacompactedmassofcells,andthethickeningisless obvious(Fig.1).Inbothcases,theemergenceofthe morphologi-callyvisibleoticplacodeisconcurrentwiththeelongationofcells
andtheacquisitionofaprominentapicobasalpolarity.Inamniotes thistakesplaceina2Dsheetofcells,whileinzebrafish,themedial oticcellsinclosecontactwiththehindbrainepithelialisebeforethe lateralcells[38,39].Whetherextrinsicsignalsemanatingfromthe surroundingtissuesdirecttheepithelialisationisunknown. Cel-lularelongationintheoticplacodeisalsoconcomitantwiththe initiationofinterkineticnuclearmigration(IKM),aprocess typi-calofneurogenicepitheliathatdescribesthedynamicoscillatory movementofnucleiwithintheelongatedcells.Inepithelia under-goingIKM, nucleiareobservedatdifferentapicobasalpositions dependentonthephaseofthecellcycle,givingthetissuea pseu-dostratified appearance[40].Prior tocelldivision, nuclei move towardstheapicalside;thecells roundupattheapicalsurface oftheepithelium,entermitosisand divide.Pseudostratification allowsgreatercelldensityoftheepithelium[41],whichhasbeen suggestedtopromoterapidtissueexpansion[42].Thisisan inter-estingpoint,sincetheoticplacodehasahighmitoticindexand itsepithelialorganisationwouldfavourarapidexpansionofthe organ.Infoetalintestinalepithelium,cellelongationand acquisi-tionofapseudostratifiedepitheliumdependonactomyosinand theactin-bindingproteinShroom3[43,44],whichisknowntobe expressedintheXenopusoticplacode[45].
Thesignalsthattriggercellstoadoptapseudostratified arrange-mentareunknown,butmostprobablyarefactorsdownstreamof theoticplacodeinducingsignals.Agoodcandidateisthe transcrip-tionfactorPax2,sinceitisexpressedinallplacodes,anditsblockade bymorpholinosinchickleadstotheabsenceofN-cadherinand N-CAM,adhesionmoleculesthatarenecessaryfortheacquisitionof columnarcellshape[46].However,othertranscriptionfactorsare
likelytoberequiredforanoticphenotype,sinceectopic expres-sionofPax2isnotsufficientforthegenerationofectopicplacodes [46].Spaltand/orSoxproteinscouldbethoseco-operativefactors. OverexpressionofSpalt4andSox3inchicknon-placodalectoderm byelectroporationiscapableofgeneratingectopicplacodaltissue [47–49].
Togetherwiththeacquisitionofcharacteristicepithelial adhe-sionpropertiessuchasadherensandtightjunctions[46,50],otic placodalcellsalsoacquireanapicobasalpolarity.Bytheotic pla-codestage,adistinctbasallaminacomposedoflaminin,fibronectin andtypeIVcollagenisalreadydepositedatthebasalsidethat sep-aratestheoticprimordiumfromtheadjacentneuraltubeinthe chick[51].Pard3,amemberoftheParcomplexinvolvedin estab-lishingapicobasalpolarity,becomesapicallylocalisedattheotic placodestageinzebrafish,togetherwiththetightjunctionprotein ZO-1[38].
2.2.3. Invaginationandhollowing
Mostorganscontainacavity,atleastduringtheinitialphases ofdevelopment,andtheinnerearisnoexception.Afterits forma-tion,theoticplacodeundergoesaseriesofmorphogeneticevents thattransforms theprimordium intoa 3D hollowedvesicle. In amniotes,theotic placodetransitsintotheoticvesiclethrough aninvaginationevent(formationoftheoticcup),whilein anam-niotes,thesphericalmassofplacodalcellsgeneratestheoticvesicle bycavitation/hollowing(seebelow).Mostprobablythereisatight connectionbetweentheestablishmentofapicobasalpolarityand oticvesicleformation,eitherbyinvaginationorothermechanisms, sinceoneofthedriversofchickoticinvaginationisF-actin, expres-sionof which becomes enhanced atthe apicaldomain ofcells justattheinvaginationstage(13somite-stage)[52].A concentra-tionofapicalactinisalsoseenjustbeforeoticplacodehollowing inthezebrafish[53].Ithasbeenproposedthatoticinvagination inthechickembryo isbiphasic. In thefirstphase, mesodermal action of FGF signalling at thebasal side of otic placodal cells inducesthephosphorylationofPLC-␥,whichinturnleadstothe activationofMyosinII.MyosinIIactivitythencausesthe depoly-merisationofbasalactinfilamentsandapicalenrichmentofthese fibres[52].Thismolecularcascadeofeventsresultsinthebasal expansionoftheoticplacode. Thesecondphase involvesapical constrictionthroughthecontractionofF-actinfibres,whichcauses cellstoadoptawedge-likeshape,triggeringtissueinvaginationas observedinmanyotherorgans[54,55].ItisplausiblethatShroom3, whichactivatesmyosinIIviaRock1andRock2andpromotes api-calconstrictionduringlensinvagination[56],playsasimilarrole duringinnerearinvagination.Duringthephaseofapical constric-tion,Myosin II,insteadof promotingbasal depolymerisationof actinfibres,drivesthecontractionofF-actinfibresattheapical side[57].Changesincompositionofthebasallaminahavealso beenproposedtobeinvolvedinoticinvaginationbymodulating theattachmentoftheoticplacodewiththeunderlyingtissue[58].It wouldbeinterestingtoexplorehowthemechanicalforcesof adja-centtissuesimpingeonoticinvagination.Inmice,Sox9hasbeen implicatedinoticinvaginationbyregulatingadhesivitybetween cellsdownstreamofEphA4,butwhetherthelackofinvaginationin
Sox9mutantswasasecondaryeffectoflossofepithelialintegrity andmechanicaldisruptionorreflectedadirectrole ofEphA4in invaginationwasnottested[59].
Inzebrafish,dynamicalimaging oftheeventsleadingtootic vesicleformationshowsthattheprocessissimilartothepreviously describedmechanismsofcordhollowinginzebrafishgutand sec-ondaryneurulationofthechickandmouseneuraltube[60–62], inwhichintercellularspaces aregeneratedattheapicalsideof cells;thecavityisnotcreatedbycellapoptosis.Intheoticplacode, twosmallcavitiesappearattheanteriorandposteriorpoles,and subsequentlyextendinanunzippingmechanismtogeneratean
elongatedcentrallumen[38].Twopolesstillformwhen antero-posteriorsignalsaredisrupted,asevidencedbytheappearanceof alumenandpositioningofhaircellsinembryoslackingFgfandHh signalling[63],butitremainstobeevaluatedwhetherthe iden-tityorpositionofthetwosmallinitiatinglumensisaffectedafter inhibitionofpatterningcues.Inchick,FGFsignallingregulatesthe constrictionnecessaryforoticinvagination[52],buttheroleofFgf signallinginotichollowinginthezebrafishisnotclear.Infgf8−/− mutantembryos,oticvesiclesaresmallerandpatterningisaffected withoutmajordefectsinlumenformation[64,65].Different pat-terningdefectsarefoundinfgf3−/− mutants[63,66],butagain, lumenformationappearstobenormal,suggestingthatinzebrafish, FgfsignallingcouldbedispensableordependentonotherFgfs.Later on,expansionofthelumenismediatedpartlybytheingression offluidcomingfromoticepithelialcells,whichshrink,remodel andactivelyparticipateintheprocessoflumengrowth[38].In chick,changesincellularvolumeduringtheexpansionphaseof theoticvesiclehavenotbeenquantified,butdorsaloticepithelial cellsundergorapidthinning[67,68](seealsobelow).Anexciting possibilityisthatdorsalthinningisaccompaniedbyfluid pump-ingintothelumen,ashappensduringtheearlystagesoflumen formationinzebrafish[38].
Inthezebrafishlaterallineprimordium,anearlystepinthe morphogenesisofsensoryepithelia(futureneuromasts)isthe for-mationofrosettesthroughapicalconstrictionofpolarisedcells. Throughtheuseofliveimagingcombinedwithgeneticand phar-macological manipulations, rosette formation was found to be dependentonFgfr-Ras-MAPKsignalling,leadingtoactivationof Rho-associatedkinase(Rock2a)andMyosinII[69].Shroom3,which isatargetofFgfsignalling,isalsorequiredforrosetteformation [70].Itwillbeinterestingtolearnwhethertheinitiallumensthat formatthepolesofthezebrafishoticvesiclesharepropertieswith themicrolumensdescribedforthelateralline,whichactto con-centratesecretedFgf,ensuringtheco-ordinatedresponseofcells withinarosettethatsharealumen[71].
Inspecieswheretheoticplacodeinvaginatesandpinchesoff fromtheoverlyingectoderm,vesicleclosurecorrelateswithafocus ofincreasedtissue apoptosis[68],but whetherblockadeofcell deathpreventsclosurehasnotbeendirectlytested.Inchick,the positionofpinchingoffseemstocorrelatewithamedio-lateral celllineageandgeneexpressionboundary(medial:Pax2;lateral:
Soho1)[72].Pax2 mutantmice,while displayinggross
morpho-geneticdefectsincochlearand semicircularcanalgrowth,have normaloticvesicleclosure,butinterestingly,inPax2;Pax8double nullmice,thereis defectiveinvaginationoftheoticcup,which remainscontinuouswiththeectoderm[73].Hereagain,Pax2/8 transcriptionfactorsemergeasgoodcandidatestocouple pattern-ingwithmorphogenesis.
3. FormationoftheVIIIthganglion:delaminationand migrationofneuroblasts
oticepithelium,neuroblastscontinueproliferatingtoexpandthe neuronalpopulationandfinallydifferentiatewithintheSAG[1,76]. Detailsofthespecificationoftheneurogenicdomainand progres-sionof otic neurogenesishave beenwellcharacterisedand are reviewedelsewhere[76–79].Herewefocusonthecellularevents leadingtothedelaminationandmigrationofoticneuroblasts.
Severalexperimentsrevealthatvestibularandauditory neu-rons of the SAG derive from distinct populations of neuronal precursorswithintheneurogenicdomainandalsoarise sequen-tially; earlybornneuroblasts constitutethevestibular ganglion andlater-bornneuronstheauditoryportionoftheSAG [80,81]. Genetictracing ofNeurog1-positivecellsin themousewas per-formedatdistincttemporalwindows;whenneuronalprecursors werelabelledatE8.5theygeneratedalmostexclusivelyvestibular neuronswhereasauditoryneuronsderivedfromneuronal precur-sors labelledat E12.5[81]. Inchick, similarresultswere found whenanalysingclonesderivedfromretrovirusinjectionsorfrom spatiallyrestrictedDiI/DiOinjections[82,83].Moreover,inthese studiesandintheworkofRaftandcolleagues,aclonalrelationship betweensensoryneuronsandhaircellsfromtheutricularmacula wasalsorevealed[84].
Inchick,delaminationoccursoveraprolongedperiodspanning fromoticcupandlateoticvesiclestages(E6),withapeakof delam-inationatstages16–17[1,83,85];inzebrafish,oticneurogenesis takesplacefrom17hpostfertilisation(hpf)until42hpf[53,86]. Unfortunately,theinformationregardingthedynamicsofcellular behavioursleadingtothedelaminationandmigrationofotic neu-roblastsisstillscarce.Itisdebatedwhetherneuroblastsdelaminate fromplacodesbyamechanismofepithelial-mesenchymal transi-tion(EMT)asshownforneuralcrestcellsexitingthedorsalneural tube.HallmarksofEMTofcranialneuralcrestareasetof transcrip-tionalprofilesthatleadtospecificcellularmorphologicalchanges, includingaswitchfromanepithelialtomesenchymalcellular phe-notypeandmigratorypropertiesreviewedin[87–90].Briefly,in chick,Bmp4andWnt1triggertheexpressionofthetranscription factorgenesSnail/Slug,Foxd3,Sox9andSox10,theproductsofwhich co-operativelyinduceaswitchinexpressionofadhesionmolecules (N-cadherintocadherin6B,7and11)andactivationofRhoB.Atthe epitheliallevel,thebasallaminaisdegraded;neuralcrestcellslose apicobasalpolarity,translocatebasallyandacquireamesenchymal phenotypeastheyexit[88].Whensomeofthesefeatureswere analysedfordelaminatingneuroblastsofdiverseplacodesinthe chick,however,neitherexpressionofSnail2,activationofRhoBnor amesenchymalphenotypewasobserved[91].Altogether,itwas concludedthatsensoryneuroblastsdonotdelaminatebyanEMT mechanism.However,ithasbecomeevidentinrecentyearsthat EMTisnotanall-or-nothingeventandintermediatetypesofEMT arepresent duringdevelopment[89]. Moreover,in chick,other transcriptionfactorssuchasTwist,ZebandE47havebeen impli-catedinEMTandthuscouldbeactinginsteadofSnail/Slug[90]. Finally,Snailexpressionisabsentinchickplacodesbutpresentin thezebrafishoticvesicleduringdelamination[65,92],reinforcing theideathatfurtherworkshouldbedonetoanalysetheparallels betweendelaminationofoticneuroblastsandneuralcrestcells.
Afterneuroblasts have emigratedfrom theepithelium, they coalesceinto a highly packed globular mass ofcells. Themost proximaldomainoftheSAG(closertotheepithelium) contains mitotically active, NeuroD-positive neuroblasts, while the dis-tal portion of the SAG contains earlier-born neurons in which divisionhasceased.Thisdistalpopulationalreadyexpresses neu-ronaldifferentiationmarkerssuchasneurofilaments,neurotrophin receptorsorIslet1[76,83,86].WavesofFGFsignalling,mediated by different FGF ligands, regulate the process of neurogenesis andSAGmaturation[86,93–95].Ithasbeenproposedthatlevels of FGFsignalling dictate theoutcome of neurogenesis.Initially, low activity of FGF signalling promotes neuroblast emergence
in theneurogenicdomain,but lateron, increasedlevelsof FGF signalling by fgf5expressedin SAG neuroblasts feedbackonto theneurogenicepitheliumtoterminateneuroblastspecification [86].Interestingly,conditionalmanipulation ofFGFsignallingin thezebrafish (usingheat shock-inducible transgenes and phar-macological interference) implicates Fgfr/PI3K/Akt signalling in zebrafishoticneurogenesis,andFgfr/Erk1/2signallinginhaircell production [95]. Othersignals suchas NGF andIgf-1 have also beenimplicatedinSAGgrowth[96–98].Buthowdoes commu-nicationbetweenotic neurons takeplaceto elicit proliferative, migratoryandstructuralchangeswithintheSAG?Cytonemes,long thinfilamentouscellularprotrusions,haverecentlybeen discov-eredascommunicationbridgesbetweencells[99].Growthfactors aretransportedalongcytonemesanddeliveredverypreciselyto neighbouringcellularmembranes[100,101].WhetherSAG neu-ronsusecytonemestoregulatetheirphysiologyormigrationis interesting but still unexplored. Anotheruntested possibility is thatasneuroblastsexittheepithelium,theyuseforces topush thepreviously-bornneuronsforward,makingthelaterstagesof neuronalmigration mainlymechanicalandpassive.Direct visu-alisation of otic neurons will elucidate the nature of neuronal migratorymovementswithintheSAGorothercellularbehaviours notconceivedoffromobservationsinfixedtissue.
Anotherinfluenceonoticneuroblastmigrationislikelytobethe neuralcrest,althoughitsimportanceappearstobedependenton speciesorplacodaltype.Inthechick,theneuralcrestestablishesa corridorforepibranchial-derivedneuroblastsduringtheir migra-tion;physicalseparationoftheneuroblastsfromthemesoderm bytheseneuralcrestcorridorshasbeensuggestedtobeessential fortheircorrectdevelopmentandaxonalgrowthtothecentral ner-voussystem[102].Similarly,blockingtheformationofneuralcrest cellprecursorsinthezebrafishwithleflunomideresultsinSAG disorganizationandaxonalbranchingdefects[103].Inthemouse, however,alossofSchwanncellsthroughconditionalknockoutof
Sox10intheneuralcrestdisruptedperipheralinnervationofthe
cochleabyspiralganglionneurons,buttheircentralprojectionsto cochlearnucleiwereunaffected[104].
4. Segregationofsensoryepitheliaandmorphogenesisof sensorychambers
Inmostorganisms,differentiationofsensoryhaircellsoccurs afteroticneurogenesis,althoughtheprocessesareconcomitant inthezebrafish.Inallspecies,however,formationoftheotic sen-sorypatchesbeginswitharelativethickeningofventralepithelium thatisaccompaniedbyaprogressivethinningofdorsalepithelium. Cellsindorsolateralregionsadoptathinsquamousmorphology; thesearedestinedtoformnon-sensoryderivatives,includingthe semicircular canal ducts (described in more detail below) and endolymphaticduct.Thethickenedventralprosensoryregion,on theotherhand,givesrisetothevarioussensoryepitheliaofthe ear,whichdifferentiateintotwomaincelltypes:sensoryhaircells, whichsitinanapicalposition,andsupportingcells,whichspan theapical-to-basalwidthoftheepitheliumandhavetheirnuclei positionedbasally(Fig.2).
Thin: precursor of semicircular canals (dorsolateral) and endolymphatic duct (dorsomedial)
Thick: prosensory (sensory-competent) domain Dorsolateral thinning
Thinning of inter-sensory patch epithelium and segregation of sensory patches
Failure of thinning and production of supernumerary hair cells (various mutant phenotypes)
Early otic vesicle
Neuroepithelium undergoing interkinetic nuclear migration
Fig.2. Generationandsegregationofsensoryepitheliaintheear.Top:schematicdiagramofageneralisedoticvesicle(lateralview).Soonafterhollowingorinvagination, thereisarelativethinningofdorsalepithelium(dependentonBMPsignallinginthechick),whereasventraloticepitheliumcontainsregionsofthickenedprosensory neuroepitheliumundergoinginterkineticnuclearmigration.Togenerateindividualandseparatesensorypatches,someregionsbecomethin,whereasothersgiveriseto differentiatedsensoryhaircells(blue)(lowerleft).Inthezebrafish,thinningcorrelateswithexpressionofE-cadherin.MutationofLmx1a,Foxg1(mouse),fgf3orjag1b (zebrafish)canresultinafailureofepithelialthinning,fusionofsensoryepitheliaand,insomecases,productionofsupernumeraryhaircells.Basedoninformationfrom referencescitedinthetext.
thatarelatersubdividedintodiscretesensorypatches[110–113]. Inzebrafish,amodelhasbeenproposedinwhichNotchsignallingis requiredtoseparatetheinitialprosensoryequivalencegroupinto twodomains,prefiguringtheutricularandsaccularmaculae[114]. Nevertheless,thiscannotbethesolemechanism,assupernumerary haircellsstillformintwodiscreteclustersinthemindbombmutant ear,inwhichNotchsignallingisdisrupted[115].Ectopichaircells candifferentiateacrosstheentireprosensoryregion,however,in zebrafishembryostreatedwithretinoicacidorwithreducedFgf signallingfromthe18–20somitestage[66].
Furthergeneticevidencefor themechanismsunderlyingthe segregationof sensoryepithelia comesfromanalysisof mutant phenotypesinwhichsensoryregionsremainundivided,although thedetailsarefarfromunderstood.Inmiceandzebrafish, macu-laearefusedorincompletelyseparatedinFgf3,Hmx3,Lmx1a,
N-myc,Otx1,Otx2andTbx1mutantsormorphants[63,66,116–124].
Thelossof interveningnon-sensorytissue betweenthesensory domainsin thesemutantscan correlatewithexpression ofthe relevantgene:expressionoftheLIM-homeodomaintranscription factorgeneLmx1a,forexample,becomesprogressivelyrestricted tonon-sensory regions, where it is thoughtto limit signalling withinorbetweensensoryepithelia[120].Separationofthe ante-riorandlateralcristaefromasingledomainisdependentonFoxg1
inthemouse:Foxg1−/−mutantsfrequentlyhaveasingleampulla sharedbybothanteriorandlateralcanalducts,whichcancontain afusedcrista[125,126].Itwillbeinterestingtoexplorewhether thedownstreamtargetsofpatterning genessuchasLmx1a and
Foxg1includegenescodingforcytoskeletalorcelladhesion
pro-teins,whichcouldmediatethecellularremodellingeventsrequired toconvertthepseudostratifiedprosensoryepitheliumintoa squa-mousnon-sensoryepithelium.
Inthezebrafish,FGFsignallingisemergingasakeyplayerin drivingtheseparationofsensorydomains.Inthefgf3−/−mutant, theutricularandsaccularmaculaeremainundivided,and super-numeraryhaircellsforminthesaccularmacula[63,66].Additional FGF ligands appear tobe required for correct formation ofthe cristae.Inthejag1b−/−mutant,bothanteriorandposteriorcristae arelost,apparentlythroughdifferentFGF-dependentmechanisms [127].Inthisstudy,MaandZhangproposethatFgf10aactsasa survivalsignalforposteriorcristasensorytissue;posteriorfgf10a
expressionandtheposteriorcrista arelostinthejag1bmutant ear.Bycontrast,azoneofFGF/ERKsignalling,possiblymediated throughFgf8a,isextendedintheanteriorprosensorydomainof
thejag1b mutant ear. Thiscorrelates withextension of a zone
Fig.3.Simplifiedschematicdiagramtocomparesemicircularcanalformationinthezebrafishorfrogearwiththatinamniotes.Onlyonecanalisillustrated;nottoscale.In fishandfrog(toprow),theproductionofextracellularmatrix(blue)helpstodriveepithelialprojectionsintotheoticlumen.Inamniotes(bottomrow),acanalpouchforms bythinningoftheoticepithelium.Celldivisioninsurroundingmesenchyme(shownas∞∞)pushesthesidesofthepouchtogether.Inallspecies,cellsadhere,fuseand resolveatafusionplate.Inthefrogandfish,thisleavesapillaroftissuespanningtheoticlumen;inthechick,cellsareclearedinthisareabyapoptosis(shownasxx).Cell clearanceleavesthecanalductformedfromtheremainingrimofepithelium.Furtherwideningofthepillar,canalgrowthandformationoftheampullaandcristaleadto thematuresemicircularcanal.
Conversely,in embryostreatedwithpharmacologicalinhibitors ofFGFsignallingorERKphosphorylation,theanteriorandlateral cristaeremainasasingleundividedzoneofthickenedepithelium containingsupernumeraryhaircells[127].Thisphenotypediffers fromthatofthefgf3mutant,wherecristaeappearrelativelynormal [63],reinforcingtheconclusionthatdifferentFGFligandsplayquite distinctrolesinoticpatterningandmorphogenesisinthezebrafish ear.
In thematureear, thesensory epithelia arenot only segre-gatedfromoneanother,butsomearealsopartitionedintodistinct sensorychambersorrecesses,connectedtotherestoftheearby narrowerforamina.Inparticular,theutriculosaccularforamen con-strictstoformaverynarrowlinkjoiningtheinferiorandsuperior partsoftheear(reviewedin[128,129]).Epithelialconstrictionto formtheseparaterecessesisdependentonthefunctionofOtx1
[121], Lmx1a[120]and N-myc[123], andcorrelateswithzones of increased cell death [130]. As theconnections betweenthe chambersnarrow,sothechambersthemselvesexpandinsize.In amniotes,thecochlearductelongatesviacellproliferation,cell–cell intercalation and convergent extension; in mammals, the duct alsocoilsintotheshell-likespiralthatgivesthecochleaitsname (reviewedin[131,132]).Pharmacologicalinhibitionexperiments usingculturedexplantsofcochlearepitheliumhavedemonstrated anautonomousroleformyosinIIindrivingconvergentextension movementsinthemousecochlea[133].Theplanarcellpolarity (PCP)pathway,mediatedbynon-canonicalWntsignalling,isalso animportantcontributortocochlearmorphogenesis.Conditional mutationofp120-cateninhasbeenshowntouncouplethe
down-streameffectorsofPCPthatmediateconvergentextensionandhair cellpolarityinthemousecochlea[134].Thisstudyalsochartsthe dynamicchangesincellularcontactsandcadherinexpressionthat occurduringdevelopmentof thecochlea[134].Anotherrecent studyhighlightstheroleofFgf10incochlearductmorphogenesis. Asinthevestibularsystem,Fgf10isexpressedincochlearsensory epithelium,whereasexpressionofitsreceptorFgfR2Bisindomains ofnon-sensorytissue.Fgf10−/− mutantshaveashorterand nar-rowercochlearductthatlacksnon-sensoryderivatives(Reissner’s membraneandtheoutersulcus)[135].
5. Morphogenesisofthesemicircularcanals
mechanismsunderlyingthegenerationof thesespecies-specific differencesarelittleknown;however,studiesofoticabnormalities inthemousestretchbackforoverfiftyyears,andsomeofthe fun-damentalstepsofsemicircularcanalmorphogenesisinthisspecies arenowreasonablywellunderstood.
Inamnioteears,thefirststepofsemicircularcanalformationis theappearanceoftwopouchesordiverticulaintheoticvesicle—a dorsalpouchthatwillgiverisetotheanteriorandposteriorcanals andcruscommune,and alateralpouchthat prefiguresthe lat-eral(horizontal)canal.Pouchformationandgrowtharedrivenby rapidthinningofthedorsolateralotocystepithelium,ratherthan alocalincreaseincellproliferation[68].Inthechick,thisthinning involvestransitionfromacolumnartoasquamouscellshape,a processthatisdependentonBMPsignalling,andcorrelateswith changesinthedistributionofE-cadherin[67].Toformthe semicir-cularcanalductsfromthepouches,thepouchsidesmovetowards eachotherandfuse:cellclearanceatthefusionplateinthe cen-treofthepouchleavesthesemicircularcanalduct,whichdevelops fromtheremainingpouchrim(Fig.3).Inthemouseear,thefirst stepin this process involvesa lossof epithelialmorphology in thepouch sides,withconcomitant disruptionof theunderlying basementmembrane[141].Itisthoughtthatcellproliferationin perioticmesenchymecontributestotheforcespushingthesides ofthefusionplatetogether;inbothNetrin1andFgf9mutants,the mitoticindexisreducedinsurroundingmesenchyme,andfusion platesfailtoform[142,143].InthezebrafishorXenopusear, for-mationofpouchesislessobviousinthesmallandcompactotic vesicle.Here,finger-likeprojectionsofepitheliumgrowtowards eachother,drivennotbymesenchymalproliferation,butby pro-ductionofextracellularmatrix[144–146].Threesuchprojections meetwithcorrespondingbulgesfromalateralprojection,where theyfusetoform three pillarsof epitheliumspanning the otic lumen.
In all species, cells at thefusion plate must recogniseeach other,touch, and fuseor intercalate. The proteins required for celladhesionatthefusionplatehavenotyetbeenidentified,but onecandidateinzebrafishistheadhesionclassGprotein-coupled receptorGpr126[145].Themurineorthologueisexpressedinthe mouseear[147],althoughitisnotyetknownwhetheritperforms asimilarroleasinzebrafish.Inthezebrafishmutant,epithelial projectionswithintheearovergrowandfailtodown-regulatethe expressionofgenescodingforavarietyofextracellularmatrix com-ponents.Celladhesionandrearrangementatthefusionplatefails, andthuspillarsandcanalductsareunabletoforminthegpr126
mutantear[145].
Oncethefusionplatehasformedinthewild-typeear,cellsare clearedfromthisarea,leavingthecanalductformedfromthe sur-roundingrimofthepouch.Inthechick,apoptosisisthoughttobe amajorcontributortocellclearance[148],whereasinthemouse, somefusionplatecellsareresorbedbackintotheductepithelium [141].ItisalsopossiblethatothercellsundergoanEMTandbecome partoftheperioticmesenchyme.Inthezebrafish,wherethefusion platesaremuchsmallerthanthoseintheamnioteear,celldeath doesnotappeartobeamajorplayer[146,149],butthedestination offusionplatecellshasnotyetbeentracedindetail.
Correctformationofthecanalductfromthepouchdependson afinebalanceofcellbehavioursatthefusionplate:toomuchcell clearance,andthecanalswillbethinortruncated;toolittle,andthe resultisanunfusedcanalpouch,remainingasanundivided vesicu-larstructure(reviewedin[150]).Inthemouseear,theextentofcell clearanceatthefusionplatecorrelateswiththedomainofNetrin1
expression,andisdependentoncross-inhibitoryinteractionswith
Lrig3toformthelateralsemicircularcanalduct[151]andwithDlx5
toformtheanteriorandposteriorsemicircularcanals[152]. Main-tenanceofDlx5expressionintherimsoftheanteriorandposterior canals(andthusprotectionfromNetrin1-dependentresorption)is
dependentonacascadeofsensory-dependentWnt,BmpandFgf signalling.Mosaicdepletionof−catenininfusionplatecellshas alsorevealedasecond,laterroleforWntsignallinginmediating resorptionatthefusionplate[152].
Onehypothesisthathasattractedmuchattentionoverthelast decade positsthat formation of thenon-sensory canal ducts is dependentonsignallingfromthedeveloping sensorycristaeto establisha‘canalgenesiszone’[131,153].Thisideaissupported byevidencefromvariousmutantphenotypes:whileampullaeand cristaecanformintheabsenceofcanalducts,examplesofnormal canalductswithoutsensorycristaearerare,andinmanymutants, bothsensoryandnon-sensorytissuesareaffectedtogether. Candi-datesignallingmoleculesthatareexpressedinthesensorydomains includethoseoftheBmp,FgfandWntfamilies,whereoneoftheir rolesistomaintainDlx5expressioninthecanalrims,asdescribed above.Bmp4,which isexpressedinthecristaeinseveral verte-bratespecies,isrequiredforthedevelopmentofbothcristaeand canaltissueinthemouseear[154].Inturn,Bmp4isrequiredfor thenormalexpressionofBmp2b;conditionallossofbmp2b func-tioninthezebrafish resultsinthelossofallthree canalducts, butcristaedeveloprelativelynormally[155].Fgf10alsomakesan interestingcasestudy:itisexpressedinthecristae,whereasthe genecodingforitsreceptor,FgfR2(IIIb),isexpressedinnon-sensory epithelium[156].Theposteriorcanalismostseverelyaffectedin murineFgf10mutants: boththecanaland itscristaaremissing inhomozygousmutants,whereasheterozygousmutantsreveala dosedependencyforFgf10,withasmallerorabsentposterior semi-circularcanal[135,157,158].Retinoicacid(RA)signallingisalso likelytobeinvolvedinsemicircularcanalmorphogenesis;inthe mouse,mutantsfortheRA-synthesisingenzymegeneRaldh3have smallandthinsemicircularcanals[159].
Not all mutant phenotypes support the canal genesis zone model,however.MicemutantforJag1orSox2,forexample,have earsthatlackoneormoreampullaeandcristae,butthecrus com-muneandcanalducts(althoughtruncated)arepresent[160–163]. Thusthecanalgenesiszonemodel,whilstprovidingauseful frame-workforunderstandingvestibularmorphogenesis,cannotaccount forallmutantphenotypes,anddatafromtheSox2andJag1mutants argueforadegreeofindependencefromsensorysignallinginthe developmentofnon-sensoryelementsofthesemicircularcanal system.
InadditiontoDlx5[164,165],mutationsinseveralother tran-scriptionfactor genesresultinsemicircularcanaldefects.These genesinclude Gbx2 [166], Hmx3(previouslyNkx5.1)[167–169],
Lmx1a[120,170]andPrx1andPrx2[171].Homozygouslossof
func-tionoftheChd7gene,whichcodesforachromatinremodelling enzyme,resultsinreducedorlossofexpressionofseveralgenes involvedinsemicircularcanalformation,includingHmx3andOtx1
(seebelow);asaresult,semicircularcanalsarelostaltogetherin
Chd7−/−mutantears[172].Interestingly,lateralcanaldefectsin
Cdh7+/− micecan berescuedbytreatmentwithaninhibitor of RetinoicAcid(RA)synthesis[173].
Table1
Semicircularcanalmutantphenotypesinthemouseandzebrafish.
Mainphenotypea Mutatedgene(s)b Species Reference
Allthreecanalducts,ampullaeandcristaevariably affectedormissing
Bmp4 Mouse [154]
Cdh7 Mouse [172]
Dlx5,Dlx5;Dlx6 Mouse [164,165,188]
Hmx2,Hmx3 Mouse [167–169]
Wnt1;Wnt3a Mouse [189]
Zic2 Mouse [190]
Allthreecanalductsmissing;ampullaeandcristaepresent bmp2b Zebrafish [155]
Partialdevelopmentofcanalductsintheabsenceofoneor moreampullaeandcristae
Jag1 Mouse [161–163]
Sox2 Mouse [160]
Anteriorandposteriorcanalsandcruscommunetruncated ormissing
Mafb Mouse [191]
Gbx2 Mouse [166]
Lateralcanalsmall,truncatedormissing Gli3 Mouse [180]
N-myc Mouse [123,124]
Otx1 Mouseandzebrafish [116–118,121]
Prx1/2 Mouse [171]
Shh Mouse [179,180]
Anteriorcanalsmallortruncated Casp3 Mouse [192]−lateralcanalfunction
alsoaffected
Posteriorcanalsmall,truncatedormissing Fgf10 Mouse [135,157,158]
Six1/Eya1compound heterozygotes
Mouse [193]
Failureordelayinfusionorcellclearanceatthefusion plate
cˇcat(ClassCand constitutivelyactiveallele)
Mouse [152]
Cdh7heterozygotes Mouse [172]
Fgf9 Mouse [143]
gpr126 Zebrafish [145]
Lmx1a Mouse [120,170]
Netrin1 Mouse [142]
Excesscellclearanceatthefusionplate;thin,truncatedor missingcanals
cˇcat(ClassA) Mouse [152]
Lrig3(lateralcanal) Mouse [151]
Thin,irregular,ordiscontinuouscanals Alk3-CKO;Alk6+/− Mouse [194]
Nor1 Mouse [195]Mutantshaveflattened
ampullae
Raldh3 Mouse [159]
Zeb1 Mouse [196]
Missing,rudimentaryorthinprojectionsorpillars (zebrafish)
hdac1 Zebrafish [197]
ptc1+/−;ptc2-/- Zebrafish [198]
sox10 Zebrafish [199]
tbx1 Zebrafish [181]
ugdh Zebrafish [200]
Overgrownprojections(zebrafish) dzip1,hip1,ptc2(ventral projectiononly)
Zebrafish [198]
gpr126 Zebrafish [145]
aPhenotypesareoftenvariable;themaindefectsaredescribedhere,butthereareoftenclassesofdifferingseverity,oradditionaldefectsmaybepresent.
bMostmutationsdescribedarehomozygousloss-of-function,butsomestudiesdescribeheterozygousphenotypesordifferentallelicvariants.Seetheindividualreferences fordetails.
thealteredexpressionofOtx1intheearsofmurineShh−/−mutants [179,180].Regulationofthesizeoftheotx1bexpressiondomainin thezebrafishoticvesicleappearstobedependentonthe oppos-ingactivitiesofFgfandRetinoicAcid(RA)signalling:Fgfpromotes, whereasRArestricts,oticotx1bexpression[66].Oticotx1b expres-sionisalsolostinzebrafishtbx1mutants[181]andsparcmorphants [182].
Althoughmanygenesrequiredforsemicircularcanalformation havebeenidentified,thechallengeisnowtolinksignalling path-waysandtranscriptionfactoractivitytospecificcellbehaviours, in orderto generatea unified model of canal formation inthe ear.By groupingtogethersimilarmutantphenotypes (Table1), andcomparingtodatafromthefrogandchick,itmaybe possi-bletoinferlinksbetweendifferentgeneproductsthatcanthen betestedexperimentally.Arecentstudyusingloss-and gain-of-functionapproaches in thechickimplicatesboth canonical and non-canonicalBMPsignallingintheregulationofDlx5andHmx3
expressioninthedorsalotocyst[183].Newcandidategeneswith rolesinoticmorphogenesisarebeingidentifiedthroughanalysis ofinsertionalmutantsin themouse[184,185] andmutagenesis screensinXenopustropicalis[186].Therearesomenotablegapsin ourunderstandingofsemicircularcanalformation:inparticular, verylittleisknownaboutformationofthedividingseptathat
delin-eatethecanalductsinthezebrafish,oroftheampullaeatthebaseof eachductthathousethecristae,bothofwhichrequirethe genera-tionofzonesofhighepithelialcurvature.Itwillbeinterestingtotest whetherthisrequiresapoptosisandmyosinII-dependentpulling forces,ashasbeenshownduringepithelialfoldinginDrosophila
[187].
6. Biomechanicsandliveimaging:convergingapproaches tounderstandmorphogenesis
3Darchitectureofmorphogenesisatthecellular,tissueororgan level.Inaddition,thephysicsofmorphogenesisisbeginningtobe elucidatedbycombiningvisualisationoffine-grainedsub-cellular detailswithnovelnon-invasive nano/picoscale technologiesfor mechanicalmanipulationoftissues.Someofthetoolsfor moni-toringmechanicsforcesarelaser-cuttingdevices,micropipettesto analysemechanical andadhesivepropertiesofcellsandtissues, and,finally,molecularforcesensors.Aplethoraofrelevantdataon theimpactofforces,tensions,pressureandflowsin embryogene-sishasbeenpublishedrecentlyfocusingongastrulation,heartand endothelialdevelopment,amongmanyothers(see[204–206]).
Theinnerear,asahighly3Dsophisticatedorganthat under-goesextensivetissueremodellingduringdevelopment,constitutes an excellent model to tackle the question of how biomechan-ics,tissuemorphogenesisandgeneregulationarecoupled.Have theadvancementsdescribedaboveimpactedonour understand-ingof inner eardevelopment? Todate, mostliveimaging data concerning inner ear development hasfocused on preplacodal movementsorplacodeformationdynamics,duetotheir accessibil-ity[20,22,23].Toourknowledge,lasermicrosurgeryexperiments toquantify mechanical forces in the inner ear have only been reportedduringlumenformationinthezebrafishinnerear[38]. There,lasercutsoftheapicalmembrane inmitoticallyrounded cellsrevealeda mechanicalroleexertedbythosecells overthe luminal membrane to expand thelumen. Further workin this directionisneededtolinkthecellulareventswiththephysical propertiesoftissuesduringoticmorphogenesis.Inaddition, light-sheetmicroscopypromisestoprovideveryinterestingdynamical data onmorphogenetic processes beyondplacode stages—such ascochlearextensionandcoiling,semicircularcanalduct forma-tionorhaircellpositioning—inthenearfuture.Theapplicationof theseimagingandphysicaltechniqueswillalsomeanthatmutant phenotypescan be exploredin new ways, leading toa deeper understandingofinnerearmorphogenesisatasystemslevel.
Acknowledgements
Work in the Whitfield lab is funded by the BBSRC (BB/M01021X/1) and work in the Alsina lab is funded by the MINNECOBFU2014-53203.
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