Apical and basal epitheliomuscular F-actin dynamics during Hydra bud evagination

44 Pages·2017·3.89 MB·English

by  Hobmayer, Engelbert

©2017.PublishedbyTheCompanyofBiologistsLtd|BiologyOpen(2017)6,1137-1148doi:10.1242/bio.022723
RESEARCHARTICLE
Apical and basal epitheliomuscular F-actin dynamics during Hydra
bud evagination
RolandAufschnaiter1,2,RolandWedlich-Sö ldner2,*,XiaomingZhang3,‡andBertHobmayer1,§
ABSTRACT shape changes in tissues are created by the coordinated activities
of individual cells including cell shape changes, cell motility,
Bending of 2D cell sheets is a fundamental morphogenetic
asymmetric cell division or changes in cell adhesion (Trinkaus,
mechanismduringanimaldevelopmentandreproduction.Acritical
1969). At the molecular level, dynamic cell behaviour is largely
player driving cell shape during tissue bending is the actin
basedontheactincytoskeletoneitherviadirectedpolymerizationof
cytoskeleton.Muchofourcurrentknowledgeaboutactindynamics
actinfilamentsorthroughinteractionofactinfilamentswithvarious
inwholeorganismsstemsfromstudiesofembryonicdevelopmentin
bindingpartnerssuchasmyosinmotors(HeisenbergandBellaiche,
bilaterian model organisms. Here, we have analyzed actin-based
2013; Guillot and Lecuit, 2013; Munjal and Lecuit, 2014). Our
processes during asexual bud evagination in the simple metazoan
understandingofdynamicactinprocessesismostadvancedinvitro
Hydra. We created transgenic Hydra strains stably expressing
inculturedcellsandintissuesofdevelopingbilaterians.Improved
the actin marker Lifeact-GFP in either ectodermal or endodermal
imaging techniques and the successful use of fluorescent markers
epitheliomuscular cells. We then combined live imaging
foractinandactin-relatedproteinshaveproventobepowerfultools
with conventional phalloidin staining to directly follow actin
todissectthecellularandbiomechanicalbasisofmorphogenesisin
reorganization. Bending of the Hydra epithelial double layer is
model organisms such as Drosophila, sea urchin, zebra fish and
initiated by a group of epitheliomuscular cells in the endodermal
Xenopus (Edwards et al., 1997; Franke et al., 2005; Burkel et al.,
layer. These cells shorten their apical-basal axis and arrange their
2007;Skoglundetal.,2008;Solonetal.,2009;Martinetal.,2009;
basalmuscleprocessesinacircularconfiguration.Weproposethat
Martin and Goldstein, 2014; Lukinavičius et al., 2014). Lifeact, a
thisrearrangementgeneratestheinitialforcestobendtheendoderm
17-amino acid actin-binding peptide from budding yeast, is a
towardstheectoderm.Convergenttissuemovementinbothepithelial
particularly promising actin-binding probe (Riedl et al., 2008). It
layerstowardsthecentreofevaginationthenleadstoelongationand
appearsnottointerferewithF-actindynamicsincellularprocesses,
extensionofthebudalongitsnewbodyaxis.Tissuemovementinto
lacks competition with endogenous actin binding proteins, and
the bud is associated with lateral intercalation of epithelial cells,
permits analysis of in vivo F-actin dynamics (Riedl et al., 2008;
remodellingofapicalseptatejunctions,andrearrangementofbasal
Spracklenetal.,2014;Lemieuxetal.,2014;DuBucetal.,2014).
muscleprocesses.Theworkpresentedhereextendstheanalysisof
Lifeact binds to actin filaments with high specificity in yeast,
morphogenetic mechanisms beyond embryonic tissues of model
filamentous fungi, plants and various metazoans. In the current
bilaterians.
study, we report the generation of stable transgenic Hydra
KEYWORDS:Lifeact,Epithelialcell,Morphogenesis,Cnidarian, expressing Lifeact-GFP and use these animals to study tissue
Tissueevagination,Evolution evaginationduringasexualreproductioninasimplemodelsystem.
HydraisamemberofthediploblasticphylumCnidaria,thesister
INTRODUCTION grouptothebilaterianclade.TheHydrapolypexhibitsonemajororal-
Bendingandfoldingofepithelialtissuesarecrucialdeterminantsin aboralbodyaxisanditsbodywall,akintothatofothercnidarians,is
creating animal shape, often initiate asexual reproduction, and formed by two epithelial layers separated by extracellular matrix
represent fundamental morphogenetic mechanisms underlying the (‘mesoglea’). The individual unit of each tissue layer is an
formationofmultipleinnerorganssuchasthegut,theneuraltube epitheliomuscular cell, which is commonly called an epithelial cell
and extremities (Gierer, 1977). The physical forces that lead to intheHydraliterature.Differentfromdevelopingepitheliainhigher
bilaterians, Hydra epithelial cells possess muscle processes located
1DepartmentforEvolutionaryDevelopmentalBiology,InstituteofZoologyand directlyadjacenttothemesoglea(Mueller,1950).Withineachlayer,
CentreforMolecularBiosciences,UniversityofInnsbruck,Technikerstr.25,A-6020 epithelialcellsareconnectedtotheirneighboursapicallyviabelt-like
Innsbruck,Austria.2Max-Planck-InstituteofBiochemistry,ResearchGroupCellular
septate junctions and basally at the level of the muscle processes
DynamicsandCellPatterning,AmKlopferspitz18,D-82152Planegg,Martinsried,
Germany.3DepartmentofAnatomyandCellBiology,UniversityofKansasMedical via multiple desmosome-like junctions. Furthermore, the basal
Centre,KansasCity,KS66160,USA. ectodermal cell membrane is connected to the mesoglea via
*Presentaddress:InstituteofCellDynamicsandImagingandCells-In-Motion
ClusterofExcellence(EXC1003–CiM),UniversityofMünster,Münster,Germany. hemidesmosome-like junctions. Ectodermal muscle processes run
‡Presentaddress:DepartmentofMolecularandCellularBiology,BaylorCollegeof along the primary oral-aboral (mouth-foot) axis of the animal,
Medicine,Houston,TX77030,USA. endodermalmuscleprocessesrunperpendiculartothisaxis.Basedon
§Authorforcorrespondence([email protected]) theirpatternsofconnectionandtheirdifferentialorientationinthetwo en
layers, muscle processes control contraction-elongation behaviour, p
B.H.,0000-0002-3401-3294 feedingandperistalticgutmovements.Athirdcellline,theinterstitial O
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttribution stemcellsystem,givesrisetonervecells,glandcells,nematocytesand gy
License(http://creativecommons.org/licenses/by/3.0),whichpermitsunrestricteduse, germcells,butcellsofthislineagearenotdirectlyinvolvedinshaping, o
distributionandreproductioninanymediumprovidedthattheoriginalworkisproperlyattributed. maintaining, or regenerating the animal’s body wall (Marcum and ol
i
Received22November2016;Accepted13June2017 Campbell,1978;SugiyamaandFujisawa,1978). B
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RESEARCHARTICLE BiologyOpen(2017)6,1137-1148doi:10.1242/bio.022723
Asexual bud formation is the primary mode of reproduction in XP_002154696) are different to each other in their predicted protein
Hydra. The body wall of an adult polyp evaginates in the lower sequence at only three amino acid positions, but are substantially
gastricregion,andtheresultingbudanlageelongatesperpendicular different at the nucleotide level. Both genes are uniformly expressed
to the mother polyp’s bodyaxis and eventually forms acomplete throughout the body column with higher mRNA levels in the
small animal that finally detaches. Throughout the entire budding ectodermallayer(Fig.S1).
process, both epithelial layers keep their unicellular conformation Inordertovisualizetheactincytoskeletoninvivo,wegenerated
intact.Budformationstartsinacircular,thickenedareainthelower transgenicpolypsexpressingLifeact-GFPunderthecontrolofthe
gastric region of the mother polyp (Otto and Campbell, 1977). Hydra actin1 promoter (Fig. 1A). Expression constructs were
Histologicalanalysisrevealedthatagroupofendodermalepithelial injectedintoembryosattheone-totwo-cellstage.Later,hatching
cells in the centre of the thickened area undergoes pronounced polypsstronglyexpressedLifeact-GFPinpatchesofectodermalor
apical-basal shortening and starts to protrude into the ectodermal endodermalepithelialcellsderivedfromembryonicprecursorcells
layer(vonGelei,1925;Philippetal.,2009).Thereafter,bothtissue that had by chance incorporated the construct into their genomic
layersevaginate.Coordinatedmovementofepithelialcellsfromall DNA. These mosaic Lifeact-GFP polyps were selected and
directions towards the evaginating centre is responsible for propagated asexually (Fig. 1B,E). They exhibited normal
transforming an initially flat sheet of parental body wall into the morphology and behaviour, and were capable of asexual and
tubular shape of an evaginating bud. This recruitment of parental sexualreproduction.Theyproducedbudswiththesamefrequency
tissueoccursuptobudstages6-7(OttoandCampbell,1977).Upto aswild-typeanimals.Hence,Lifeact-GFPexpressiondidnotseem
this phase, enhanced cell proliferation and oriented cell division to critically interferewith major physiological and developmental
havebeenshowntoplaynoroleinshapingthebud(Clarksonand processes(see Discussion). Todetect whether Lifeact-GFP fusion
Wolpert, 1967; Websterand Hamilton, 1972; Otto and Campbell, proteinsspecificallylocalizedtotheactincytoskeleton,transgenic
1977; Holstein et al., 1991; Shimizu et al., 1995). Only in later animalswerefixedandcounterstainedwithrhodamine-phalloidin.
budding stages doestissue growth contribute to maturation of the Inthesepreparations,wefoundcompleteoverlapofthetwolabelsat
bud (Otto and Campbell, 1977). Ectodermal epithelial cells have thesiteofactin-basedstructures(Fig.S2).
been shown to intercalate laterally during budding (Philipp et al.,
2009),similartoconvergentextensionmovementsofmesenchymal ArchitectureoftheactincytoskeletoninHydraepithelial
cells in Xenopus or germ band elongation in Drosophila (Keller cells
et al., 2000; Rauzi et al., 2008). It has been speculated that basal MosaicanimalswithonlyfewLifeact-GFPpositivecellsprovedto
muscle processes act in epithelial cell motility and bud beparticularlywellsuitedforobservationsofsinglecellsinintact
morphogenesis,butthehistologicalmethodsusedtostainactinor tissue.Inthesepolyps,theF-actincytoskeletoncouldbevisualized
othercytoskeletalelementsofferedonlylimitedresolutionanddid atunprecedentedresolution.Asobservedearlier,muscleprocesses
notallowliveimaging(Otto,1977;Campbell,1980). atthebaseofepithelialcellsrepresentedthemostprominentactin
Although Hydra bud formation has been intensively studied in structures. In the ectoderm, they were organized in discrete,
the past, core issues remain unanswered. The behaviour of single condensed fibres running along the oral-aboral axis of the polyp
epithelial cells during tissue evagination is not well understood. (Fig. 1C,H). Their length was variable (Fig. 1C) and ranged
Furthermore,thecontributionofeachofthetwoepitheliallayersto between one and five times the planar epithelial cell diameter as
thebuddingprocessandthedetaileddynamicsofactinstructuresas measured at the apical surface. One ectodermal epithelial cell
operatorsofthedeveloping3Dstructureareunresolved.Inorderto possessedonaveragetwotothreebasalmuscleprocesses(2.4±0.6,
approachthesetopics,weaimedatdevelopingawaytovisualizethe n=52cells)(Fig.1C),consistentwithearlierobservationsmadeby
actincytoskeletoninvivoinHydraepithelialcells,totrackdynamic electronmicroscopy(Mueller,1950).
changesofactinnetworksduringtissuebendingandmovementinto Intheapicalpartofectodermalepithelialcells,Lifeact-GFPwas
evaginating buds, and to deduce from these observations possible concentrated in a circumferential belt of actin filaments at the
underlying forces that drive morphogenesis. We created two position of septate junctions (Fig. 1D,H). In fixed, phalloidin-
transgenic Hydra strains, in which Lifeact-GFP localized labelled specimens, this structure resembled a chain-like
specifically to actin filaments in ectodermal and endodermal arrangement (Fig. 1I). In living Lifeact-GFP cells (Fig. 1J),
epithelial cells, revealing previously undescribed F-actin however, it appeared as a continuous belt indicating that the
structures. In vivo tracking of Lifeact-GFP combined with chain-likearrangementisanartefactcausedbychemicalfixation.In
phalloidin staining on fixed samples revealed distinct patterns of addition,afinenetworkofcorticalactinfilamentsstretchedacross
reorientationofmuscleprocesses,remodellingofactin-networksat the width of the cell under the apical membrane (Fig. 1J,
apical septate junctions, and the formation of muscle process-like arrowheads). High resolution live imaging using total internal
structures associated with motility. These newly generated reflection fluorescence (TIRF) microscopy revealed continuous
transgenic animal strains have the potential to provide an movement within this cortical actin network (Movie 1). TIRF
understanding of cytoskeletal dynamics not only during bud microscopy also revealed filopodia-like protrusions extending
morphogenesis, but also during the establishment of epithelial laterally at the most apical region (arrows in Fig. 1K; Movie 2).
polarityandregeneration. These protrusions appear to represent the cytoplasmic extensions
frequently found in transmission electron microscopic images
n
RESULTS connectingneighbouringcellsviaseptatejunctions(Fig.S3). e
TransgenicHydraexpressingLifeact-GFP Muscleprocessesatthebasalfaceofendodermalepithelialcells p
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The Hydra genome encodes several actin and actin-like were oriented perpendicular to the oral-aboral axis of the polyp.
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genes(XM_002154426,XM_002154660,XM_012700059,XM_ Theyoccurredinmuchlargernumbers(commonly10ormoreina g
002158909,XM_002158614/Sc4wPfr_228.g20688,XM_002160174/ relaxed cell), covered the entire basal surface, and did not extend o
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Sc4wPfr_422.g20281). The two most conserved actin genes significantly beyond the periphery of the cell (Fig. 1F,H). In o
i
(named here actin1: XP_002154462, Fisherand Bode, 1989; actin2: phalloidin-stainedpreparations,endodermalmuscleprocesseswere B
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Fig.1.TransgenicHydrastrainsexpressing
Lifeact-GFP.(A)Codon-optimizedsequenceofthe
lifeact-GFPexpressionconstructdrivenbythe
Hydraactin1promoter.(B)Hydrapolypwithmosaic
expressionofLifeact-GFPinlargeareasofthe
ectodermallayer.(C)Clusterofthreetransgenic
ectodermalepithelialcellswithfocuslevelatthe
basalmuscleprocessesrunningalongtheprimary
oral-aboralaxis;(D)thesamecellclusterwithfocus
atapicalcelljunctions.(E)Hydrapolypwithmosaic
expressionofLifeact-GFPintheendodermallayer.
(F)Clusteroffourtransgenicendodermalepithelial
cellswithfocuslevelonbasalmuscleprocesses;
(G)thesamecellclusterwithfocusatapicalcell
junctions.(H)Schematicdrawingofanectodermal
andanendodermalepithelialcellaspositionedin
thebodywallofHydrawithprominentactin
structureshighlightedingreen.Basalmuscle
processesinectodermalandendodermalepithelial
cellsareorientedperpendiculartoeachother.The
mesoglealinkingbothtissuelayersisnotincluded.
Theconnectionsbetweenectodermaland
endodermalepithelialcellsacrossthemesogleaare
alsonotshown.(I-K)F-actinstructuresattheapical
surfaceofectodermalepithelialcells.(I)Confocal
projection(3.5µmdepth)atapicalcelljunctionsin
fixedtissuestainedwithAlexaFluor488Phalloidin.
(J)Correspondingconfocalsectionatapicalcell
junctionsinalivingmosaicLifeact-GFPpolyp.
(K)Actin-basedapicolaterallamellipodia-like
structures(arrows)asvisualizedinasingleframeof
amovietakenwithaTIRFmicroscopeinaliving
mosaicLifeact-GFPpolyp(seeMovie2).Theblack
spacearoundthetransgeniccellisoccupiedby
nontransgenicepithelialcells.Scalebars:50µmin
C,D,FandG;10µminI-K.
obscured by the much brighter ectodermal fibres (Fig. S2). As in parentandbud.Muscleprocessesofcellsenteringthebudatlateral
the ectoderm, we found Lifeact-GFP-labelled actin rings at the positionshavetoreorientbyupto90°withrespecttotheirfuture
sites of endodermal septate junctions (Fig. 1G,H). However, it orientationinthebud(Otto,1977).
was not possible to study the apical endodermal region using Budinitiationstartswithathickeningofbothepitheliallayersina
confocalorTIRFmicroscopyduetothethicknessoftheepithelial circularareainthelowerbodycolumn(Fig.S4A-D).Endodermal
double layer. muscle processes appeared more condensed in the centre of the
thickenedareainlatestage1buds,andtheystartedtodeviatefrom
Initiationofbudformationinvolvesreorientationof theregulararrangementbyforming aneye-shapedarrayplacedin
endodermalmuscleprocesses the centre (Fig. 2A,B). These changes occurred before the basal
Theoral-aboralaxisofthedevelopingbudisformedatarightangle muscle layers associated with the mesoglea exhibited any visible
to the axis of the parental polyp. As a consequence, muscle curvature(Fig.2A′,B′).Shortlyafter,instage2buds,endodermal n
e
processesinboththeectodermandendodermhavetoreorganizein muscle processes established a circular arrangement (Fig. 2C), p
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order to establish normal contractile behaviour in the bud. The which coincided with an endodermal bending towards the
degreeofmuscleprocessreorientationinaparticularcelldepends ectodermal layer (Fig. 2C′,D). Ectodermal epithelial cells lying gy
onitspositionwithinthebud.Whencellsoftheparentalpolypenter directly over these endodermal cells shortened their apical-basal o
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thebudontheupperorlowerside(facingtheheadorfootofthe diameter (Fig. S5A-C) and, as a result, the outer surface of the o
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motherpolyp),theirmuscleprocessesareproperlyorientedinboth tissue bilayer did not show obvious curvature. From stage 3 on, B
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Fig.2.Earlycirculararrangementofendodermalmuscleprocesses.(A,B,C)Confocalprojectionsofthebasalregionsofectodermalandendodermal
epithelialcellsofthebuddingzonestainedwithAlexaFluor488Phalloidin.(A)Latestage1bud,inwhichectodermalmuscleprocessesrunverticallyand
endodermalonesrunhorizontally.Inthecentre,endodermalmuscleprocessesstarttodeviatefromtheirregularorientationandappearshortened.(B)Eye-
shapedarrangementofendodermalmuscleprocessesinbudstage1-2.(C)Circularorientationofendodermalmuscleprocessesinbudstage2,which
reflectsthecorrectendodermalplanarpolaritywithinthenewlyformingpolyp.(A′,B′,C′)Orthogonalopticalsectionsof(A,B,C)atthepositionsindicatedbyarrows
showthatendodermalbendingintotheectodermallayeriscorrelatedwithcirculararrangementofendodermalmuscleprocesses.(D)Schemeofthe
arrangementofendodermalmuscleprocessesatbudstage2.ENML,endodermalmusclelayer;M,mesoglea;EC,ectodermallayer.(E)Timingofendodermal
muscleprocessreorientationaccordingtobudstagesfromOttoandCampbell(1977).Depthofconfocalprojections:20µminA;15µminB;25µmin
C.Scalebars:50µminAandB;20µminC.
evagination proceeded in both epithelial layers to form a shortenedduringreorientationandelongatedtooriginallengthafter
macroscopicallyvisiblebudprotrusion(Fig.S5D). reorientation(Fig.4A′,B′,C′,D).
Ectodermal muscle processes maintained their original Endodermal muscle processes showed basically the same
orientationuntilbudstage2,whentheywerestillalignedasinthe behaviour (Fig. 5). Cells located on the lower side of an
adjacent parental tissue. The distance between them, however, evaginating bud turned them clockwise, cells of the upper side
increased as aresult of thecurvatureof the developing protrusion counter-clockwise (Fig. 5A′,B′,C). Changes in muscle process
(Fig.3A)(Otto,1977).Startingatbudstage3,ectodermalmuscle length,however,werenotobservedinendodermalcells.
processes began to reorient their polarity until they established a
bud-specificaxialalignment(Fig.3B).Atriangularareawasvisible Lateralintercalationisaccompaniedbypolarizedjunctional
atthebaseofstage3andolderbuds,wheremuscleprocessesaligned remodelling
paralleltotheparentalaxiswereseparatedfromthosealigningwith We also studied junctional remodelling in ectodermal epithelial
thenewlyformedaxisofthebud(Fig.3B,B′)(Otto,1977). cells by tracking the relative positions of the apical actin belts
connectedtoseptatejunctions(Fig.6).Duringlateralintercalation,
Reorientationofmuscleprocessesduringtissue apical contacts were established between formerly unconnected
recruitment cellsalongthebud’sneworal-aboralaxis(Fig.6A′,B′,C′,D,yellow
The fast outgrowth of the bud between stages 3 and 6 is due to dots). Furthermore, formerly connected cells detached in an
recruitment of parental tissue by roughly concentric rings of orientation perpendicular to this axis (Fig. 6A′,B′,C′,D, red
epithelialcellsmovingtowardsthecentreofevagination(Ottoand circles; Movie 3). This intercalation behaviour resulted in an
Campbell, 1977). Due to this movement pattern, individual extension of the tissue along the bud’s axis (Fig. 6A′,B′,C′).
epithelial cells have to rearrange relative to each other by lateral Junctionalremodellingoccurredinalargeareacoveringtheparental
intercalation (Otto and Campbell, 1977; Philipp et al., 2009). In tissueandtheevaginatingbud,butwasnotobservedoutsideofthe
order to track the behaviour of actin structures during lateral buddingzone(Fig.6E).
intercalation, we followed changes in muscle process orientation Asintheectoderm,recruitmentofendodermaltissueintothebud
in vivo using Lifeact-GFP transgenic animals. Ectodermal muscle requires rearrangement of epithelial cells relative to each other.
n
processes moving into the bud laterally reoriented in a distinct Here,microscopicresolutionwaslimitedandourobservationsdeep
e
spatial pattern (Fig. 4). Cells located in the lower side of the intheHydraendodermaltissuelayercouldnotresolvechangesin p
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evaginating bud (oriented towards the parental foot) turned their apical actin belts at the single cell level. Nevertheless, we did
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muscle processes clockwise; cells in the upper side (oriented observe clear changes in Lifeact-GFP-positive endodermal cell g
towardstheparentalhead)turnedthemcounter-clockwise(Fig.4A′, clusters. As in the ectoderm, they narrowed along the axis of the o
B′,C′,D). In addition to a change in direction, muscle processes parentalpolypandextendedalongthenewlyformedbudaxisdueto ol
i
exhibited a transient reduction in length upon reorientation: they lateralintercalationofadjacentcells(Fig.5). B
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Fig.3.Lateralviewsofearlybudstagesshowingectodermalmuscleprocessreorientation.(A,B)ConfocalprojectionsofspecimensstainedwithAlexa
Fluor488Phalloidin.(A′,B′)Schematicdrawingsofectodermalmuscleprocesses(dashedlines)atthecorrespondingstages.Whileectodermalmuscle
processesmovingintothebudfromoralandaboraldirectionsareorientedrathercorrectlywithrespecttothebudsnewbodyaxes,thoseenteringthebud
fromlateralareashavetoincreasinglyrearrangeinordertoattainproperorientation.Thisresultsinatriangleshapeatbudstage3-4.(C)Timingofectodermal
muscleprocessreorientationaccordingtobudstagesfromOttoandCampbell(1977).Projectiondepths:30µminA;10µminB.ECML,ectodermalmuscle
layer;ES,ectodermalapicalsurface.Scalebars:50µm.
Ectopicectodermalmuscleprocessesareassociatedwith frequency as wild-type animals. We found identical F-actin
cellmotility dynamics at specific budding stages in Lifeact-GFP animals and
Duringtissuerecruitment,individualectodermalmuscleprocesses phalloidin-stained wild-type preparations, indicating that the
not only reoriented, but displayed highly irregular polarity and presence of Lifeact-GFP did not noticeably affect the behaviour
spatialarrangements(Fig.7).Thesedetachedfibreswerefoundin of actin filaments. Bud formation, however, occurred at a slightly
evaginating bud tissue, but also at positions outside of the lowerrateinfullytransgenicpolyps.Weobservedthatthesepolyps
evaginating tissue in the region between bud and parental polyp catchandeatsmallernumbersofbrineshrimpperdayascompared
(Fig. 7A-C). They werenot presentin morphogenetically inactive towild-typepolyps.Thiscouldbeduetoamarginalinterferenceof
regionsofthepolyp(Fig.7D).Highmagnificationlivetrackingof Lifeact-GFP with actin contractility involved in nematocyte
individualectodermalcellsrevealedthatthesubcellularlocalization dischargeormovementoftentacles.
ofdetachedmuscleprocesseswasdynamicallyremodelledintime
intervalsofafewhourssupportingaviewthattheycontributetocell EpithelialF-actinarchitectureintransgenicLifeact-GFP
movement(Fig.7E-H).Inaddition,theyextendedinanapical-basal Hydra
direction upto theapicalactin belt (Fig.7E). Thereforetheymay Ectodermal and endodermal epithelial cells are surrounded by an
alsobeinvolvedincoordinatingapicalandbasalpartsofamoving apical F-actin belt associated with septate junctions. Septate
epithelialcell. junctionsperform twomajorfunctionsinHydra:restricting trans-
cellular permeabilityand mediating intercellularadhesion (Wood,
DISCUSSION 1985). Linking F-actin to septate junctions is likely to provide
Dynamics of actomyosin networks underlie many morphogenetic stability against mechanical forces and to connect actin networks
cell behaviours (Guillot and Lecuit, 2013; Heisenberg and betweenneighbouringcellsinordertotransmitforceattheapical
Bellaiche, 2013; Munjal and Lecuit, 2014). Therefore, planeoftheepithelium.Inectodermalepithelialcells,wedetecteda
considerable effort has been made to develop methods for live finenetworkofsmalleractinfilamentsjustbelowtheapicalsurface
imagingofF-actin.InordertospecificallylabelF-actinnetworksat connectingtheF-actinbeltacrosstheentireplanarcelldiameter.The
highest resolution, we produced stable transgenic Hydra polyps function of this network has not yet been explored, but an
expressing Lifeact-GFP in ectodermal and endodermal epithelial involvement in the control of apical cell shape seems likely. In
n
cell lineages. Our approach follows previous attempts to create addition,wefounddynamicactin-filledfilopodiaextendingoutward
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whole animal Lifeact models to studyorganismic physiologyand fromtheapicalF-actinbelt,whichmayengageintheformationand p
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development (Riedl et al., 2010; Phng et al., 2013). We have re-organizationofcellcontactsitesbetweenneighbouringcells.
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cultivatedtheseLifeact-GFPstrainsforseveralyearsandhavenot By enhancing the resolution of earlier studies, which had used g
observed any significant impairment of polyp behaviour, maceration preparation and ultrathin serial sectioning (Mueller, o
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reproduction, or regeneration. The mosaic Lifeact-GFP polyps 1950; David, 1973; West, 1978), Lifeact-GFP-expressing o
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usedinthisstudyproducebudswiththesamestagingpatternand ectodermal epithelial cells show two to three long, 1-2µm thick B
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Fig.4.Reorientationofectodermal
muscleprocessesduringbud
evagination.(A,B,C)Liveimagingofa
developingbudatthreetimepoints(0,4,8h)
showingmuscleprocessesinpatchesof
transgeniccells.(A′,B′,C′)Magnifiedimages
revealtherotationaldirectionandcontraction
duringreorientationintheupper(oriented
towardstheheadofthemotherpolyp)and
lower(orientedtowardsthefootofthemother
polyp)halfofthebud.Starsandtriangles
labeltwoindividualmuscleprocessesin
ordertodemonstrateconvergingmovement.
(D)Schematicsummaryoftheevents.Scale
bars:200µminA,BandC;100µminA′,B′
andC′.
muscleprocesses.Endodermalepithelialcells,incontrast,exhibita byWebsterandHamilton(1972)andGrafandGierer(1980).Inthe
highernumberofshorterandmuchfinermuscleprocessescovering planula larva of the anthozoan Nematostella vectensis, Fritz et al.
the entire basal surface. At the ultrastructural level, ectodermal (2013) recently showed that tentacle evagination also starts in a
and endodermal muscle processes have been demonstrated to be placode-likestructureofthickenedectodermalepithelialcells.von
similarlyconstructed,containingfilamentswiththesamediameter Gelei (1925) further described a distinct behaviour in a group of
(Haynes et al., 1968). Thus, a different degree of actin fibre 10-20endodermalepithelialcellsinthecentreofthethickenedarea,
condensationbetweenectodermandendodermisprobablybasedon which substantially decreased their apical-basal diameter and
differentialuseofscaffoldandlinkerproteins. started to bend towards the ectodermal layer. Based on this, he
postulatedthatendodermalpressuretowardstheectodermprovided
Actindynamicsduringtissuebending the initial mechanical force for bending. Campbell (1967)
Hydra bud formation can be separated into three phases with speculated that these cell shape changes in the endoderm result
presumably phase-specific morphogenetic processes: initiation, fromadecreaseinlateralaffinitybetweenthecellsandanincrease
elongation, and detachment. Gierer (1977) argued that a 2D cell inaffinityfortheextracellularmatrix,whichwouldcreateabending
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sheet such as the gastric body wall of Hydra is in a state of momentintheendodermtowardstheectoderm.Notably,Sherrard
e
biomechanical stability, so that a distinct mechanical force etal.(2010)foundthatapical-basalshorteningofendodermalcells p
(‘bendingmoment’)isrequiredtobreaktheflatstateandtocause drivesinvaginationduringCionagastrulation,andinthiscasethe O
y
curvature. von Gelei (1925) was the first to provide a detailed observed cell shape changes were clearly caused by actomyosin g
histological description of the earliest bud stages. He described contractility. o
l
thickening of the ecto- and endoderm in the prospective budding In addition to apical-basal shape changes, Otto (1977) o
i
areatoformaflat,placode-likestructure,whichwaslaterconfirmed conjectured that contraction of ring-shaped endodermal muscle B
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RESEARCHARTICLE BiologyOpen(2017)6,1137-1148doi:10.1242/bio.022723
Fig.5.Reorientationofendodermalmuscle
processesduringbudevagination.(A,B)Live
imagingofadevelopingbudattwotimepoints
(0,6h)showingmuscleprocessesinpatchesof
transgeniccells.(A′,B′)Magnifiedimagesrevealthe
rotationaldirectionduringreorientationintheupper
(orientedtowardstheheadofthemotherpolyp)and
lower(orientedtowardsthefootofthemotherpolyp)
halfofthebud.(C)Schematicsummaryofthe
events.Scalebars:200µminAandB;100µmin
A′andB′.
processes may contribute to a displacement of the endoderm responseswithstrongestexpressionofthephenotypeinthecentreof
towards the ectoderm, analogous to the elongation of the body theplacode(Fig.S4A-D;vonGelei,1925;Philippetal.,2009).This
column by contraction of endodermal muscle processes. The data centre will later develop into the evaginating tip, and we propose
presentedhereandinourpreviousstudy(Philippetal.,2009)agree thatitmayactasapointsourceforinstructivesignals.Inapreceding
surprisingly well with this model. Contraction of an endodermal study (Philipp et al., 2009), we suggested that secreted Wnt5
muscleprocessringformedbybudstage2couldpromotetheinitial proteins may induce changes in shape and polarity in adjacent
bending of the endodermal layer (Fig. 8A). Notably, endodermal endodermalcells.wnt5isexpressedinasmallgroupofectodermal
muscle process rings are also formed, when tentacles start to epithelial cells located directly above the responding endodermal
n
evaginate in developing buds and during head regeneration (Fig. cells,andwnt5activationisdirectlycorrelatedwiththeinductionof
e
S4E-G),andwhenectopictentaclesstarttoappearalongthebody tissueevaginationduringbudandtentacleformation.Furthermore, p
O
column in alsterpaullone-treated polyps (Philipp et al., 2009; Wnt/PCP signalling is a major source of polarity cuesthroughout
y
Anton-Erxlebenetal.,2009). bilateriandevelopmentalmodels(Keller,2005;Zallen,2007;Gros g
What are the molecular signals underlying these changes in et al., 2009). Notably, transcriptional activation of wnt5 occurs in o
l
cellular behaviour? Thickening in both layers of the bud placode ectodermalepithelialcellsjustpriortothefirstmorphologicalsigns o
i
and apical-basal shape changes in endodermal cells are graded oftissuebending,whilechangesincellshapeandcellpolarityare B
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RESEARCHARTICLE BiologyOpen(2017)6,1137-1148doi:10.1242/bio.022723
Fig.6.Polarizedremodellingof
ectodermalapicalcelljunctionsduring
budevagination.(A,B,C)Liveimagingofa
developingbudatthreetimepoints(0,4,8h)
showingalargerpatchoftransgeniccells.
(A′,B′,C′)Magnifiedimagesarefocusedon
F-actinassociatedwithapicalseptate
junctions;individualtransgeniccellsare
labelledwithnumbers.(D)Schematic
drawingsofthetransgeniccellsinA′,B′and
C′,highlightingtheirpositionsandcontact
areas.Yellowdotsmarknewcell-cell
contacts,whichareestablishedalongthe
newlydevelopingoral-aboralaxisofthebud.
Redcirclesmarkdetachmentofexistingcell-
cellcontacts,orientedperpendiculartothe
oral-aboralaxisofthebud.(E)Relativerate
ofapicaljunctionalremodellinginthe
evaginatingarea(greybar)ascompared
withcontroltissueinthemid-gastricregion
(whitebar).Todeterminetherateof
remodelling,thenumberofpolarized
changes(yellowdots+redcircles)per
existingcell-cellcontactbetweentwo
transgenicectodermalepithelialcellswas
countedduringaperiodof12h.For
evaginatingcells,atotalofsixcellclusters
including110cell-cellcontactswere
analyzed.Duringthetrackingperiod,these
cellsmovedfromroughly−200µmdistance
tothebud-parentborderwithinthemother
polyptoroughly300µmdistancetothebud-
parentborderinthebud,andaboutoneout
oftwocellseitherconnectedtoanewcell
neighbourorlostcontacttoitsprevious
neighbour.Forcontrolcells,atotalofthree
cellclustersincluding157cell-cellcontacts
wereanalyzed.Thesecellsshowedmuch
slowermovementalongtheoral-aboralaxis
ofthemotherpolyp,andchangedtheir
neighboursataverylowrate.Scalebars:
200µminA,BandC;100µminA′,B′andC
′.
first observed in the endodermal layer. Such clear functional convergentextension-likemanneralongaroughlycircularfatemap:
partitioning along the outside-inside direction of the epithelial cellslocatedneartheevaginatingcentrewillendupintheoral/distal
bilayer ensures correct bending (Gierer, 1977). In fact, bud part of the bud; those located more distantly will move to a more
invaginationhasneverbeenobservedinHydra.Bendingofabud aboral/proximal part of the bud (Otto and Campbell, 1977). The
always occurs in the correct outward direction, whereas single- mechanismsgeneratingthismovementareunclear.Duringsteady-
layered systems such asthe seaurchin gastrula appear to be more state tissue turnover, epithelial cells in the mid-gastric region are
error-prone and can sometimes bend in the wrong direction and displaced together with the underlying mesoglea along the oral-
n
hence produce exogastrula phenotypes (Gustafson and Wolpert, aboral axis towards the foot (Aufschnaiter et al., 2011). With the
e
1963). initiationofabud,however,cellsinthebuddingzonechangetheir p
O
directionandstarttomovefromallsidestowardsthefuturecentreof
y
Actindynamicsduringtissuerecruitment evagination. The mechanisms generating this movement are g
Elongation of the early bud is driven by recruitment of epithelial unclear. Campbell (1980) proposed a hypothesis that epithelial o
l
tissue from the mother polyp into the newly forming protrusion cells actively crawl on the mesoglea by using their contractile o
i
(Fig.8B).Epithelialcellsexhibitlateralintercalationandmoveina muscle processes. Recent support for this view came from B
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RESEARCHARTICLE BiologyOpen(2017)6,1137-1148doi:10.1242/bio.022723
Fig.7.Randomlyorientedectodermal
muscleprocessesinthelargerbudding
area.(A)Schematicshowingtheperspective
oftheobserverontheplaneoftheepithelium
alonganoutside-insidedirection.Confocal
projectionforB-Dstartsdirectlybelowthe
apicalseptatejunctionsofectodermal
epithelialcellsandextendstowardsthe
basalendofendodermalepithelialcells
includingtheirmusclelayer.Sampleswere
stainedwithAlexaFluor488Phalloidin.
(B)Confocalprojectionofastage2-3bud
andthesurroundingtissueshowingalarge
numberofmuscleprocessesbentor
randomlyoriented.Xmarksthecentreof
evagination;thedottedcircleindicatesthe
bud-parentborder.(C)Magnifiedviewas
indicatedinB.(D)Regulararrangementof
muscleprocessesinunevaginatedcontrol
tissuefromthemid-gastricregion.
(E-H)Shortintervallivetrackingofan
individualectodermalepithelialcellduring
tissuerecruitmentshowsdynamic
remodellingofitsactinfibers.Scalebars:
100µminB;20µminCandD;100µm
inE-H.
observations of epithelial cells moving into the bud relative to Lateral cell intercalation has been thoroughly investigated in
the underlying mesoglea (Aufschnaiter et al., 2011). Possible embryonicepitheliaofvariousbilaterianswithaparticularfocuson
‘molecularclutch’mechanismsofintegrin-basedforcetransmission apical(Drosophila)andlateral(vertebrates)actindynamics(Munjal
to an extracellular matrix have been reviewed recently (Case and and Lecuit, 2014). The data presented here, however, provide
Waterman, 2015). Here, we show that indeed a large fraction of evidence that a force for morphogenetic movement may be
ectodermalmuscleprocessesinthebuddingareaisdetachedfrom generatedinthebasalpartoftheepitheliumbycontractilemuscle
themesoglea anddisconnected from neighbouringcells.Theyare processes.Similarphenomenahaveactuallybeendescribedinrare
n
presentatanyradialpositioninthebudrulingoutthattheyrepresent cases,duringepithelialcellrearrangementinthedorsalhypodermis
e
axially reorienting muscle processes. Furthermore, they remodel ofCaenorhabditiselegans(Williams-Massonetal.,1998),during p
O
within short time intervals. An engagement of such detached convergent extension of the notochord in ascidians (Munro and
y
muscle processes in cell motility is further supported by their Odell,2002),andduringconvergentextensionofthemouseneural g
presence in evaginating tentacles and during head regeneration, plate(Williamsetal.,2014).Inalltheseexamples,epithelialcells o
l
two other processes involving cell migration (R.A. and B.H., formactin-basedprotrusionsatabasolateralposition,andthisbasal o
i
unpublisheddata). protrusiveactivityclearlyactsinintercalationmovements.Despite B
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RESEARCHARTICLE BiologyOpen(2017)6,1137-1148doi:10.1242/bio.022723
Fig.8.Actin-basedprocessesinvolvedin
budmorphogenesis.(A)Bending.Circular
arrangementandcontractileactivityofbasal
muscleprocessesinagroupofendodermal
epithelialcellsmayparticipateininitiating
tissuebending.Thebluelineinthelower
schemerepresentsthemesoglea,andthe
ectodermallayerisnotshown.Theconfocal
imageonastage2budistakenfromPhilipp
etal.(2009)withpermissionfromPNAS.
(B)Tissuerecruitment.Movementof
epithelialcellstowardsthecentreof
evaginationduringbudelongationandtissue
recruitmentinvolvesapicaljunction
remodellingandretraction,re-polarization
andre-attachmentontothemesogleaof
basalmuscleprocesses.
obvious structural differences between basolateral, filopodia-like oral-aboral polyp body axis (Seybold et al., 2016; Livshits et al.,
protrusions and basal muscle processes, they emphasize a 2017). Hence, the head organizeras primary signalling centre for
potentially underestimated and more general role of actin axial patterning may be responsible for providing this directional
dynamics in the basal compartment of epithelial and cueinHydraaggregatesandcoulddosoduringbuddevelopment.
epitheliomuscularcellsfortissuemorphogenesis. Finally, cells changing their positions relative to each other by
Wetriedtointerferewithcytoskeletaldynamicsbyusingasetof lateral intercalation need to continuously modify their apical
actinandmyosininhibitorsincludingCytochalasinD,Latrunculin, junctional complexes in order to maintain the stability, integrity
SwinholideandBlebbistatin.However,whenpolypswithastage1- and permeability of a cellular sheet. We observed epithelial
2 bud were treated with inhibitor, none of them implemented junctional remodelling during tissue recruitment, and its spatial
specific inhibition of bud initiation or tissue recruitment. It was dynamicscorrespondstothepatterndescribedinbilateriantissues
difficult to assess the effect of the myosin inhibitor Blebbistatin, (Fig.8B).Itshouldbenotedthatbilaterianjunctionalremodelling
because it totally blocked contractile behaviour in the polyp and hasbeenstudiedingreatdetailincadherin-basedadherensjunctions
halted any tissue movement including budding and early head (Takeichi, 2014). In Hydra, however, these apical complexes are
regeneration(Fig.S6A).Amongtheactininhibitors,CytochalasinD representedbyseptatejunctions,thestructureofwhichiscurrently
strongly blocked wound healing during early head regeneration notpreciselyknown.Preliminarydataemphasizeco-localizationof
(Fig. S6B), but had no obvious effect on bud evagination in the beta-catenin and other members of the cadherin-catenin-adhesion
regularconcentrationrangeofupto25µM.Weinterpretthisresult complexatHydraseptatejunctions(Brounetal.,2005;S.Pontasch
as an inabilityof CytochalasinD to interfere with existing muscle andB.H.,unpublisheddata),butalsorevealthepresenceofclaudin
processesandapicalactinbeltsintheevaginatingbudtissue. (M.-K.EderandB.H.,unpublisheddata).Duetothesedifferences
The epithelial movement pattern observed during tissue in structure and composition, apical remodelling of septate and
recruitment clearly needs a directional cue, which is undefined at adherencejunctionsmustusedifferentmolecularmechanisms.
present,butwhichwepresumeisanattractivesignallocatedatthe
evaginatingcentre.Candidatesforsuchasignal,whicharedefined Conclusion
bytheirlocalexpressionintheevaginatingcentreofayoungbud In- and evagination of tissues are fundamental mechanisms to
andbytheirknownroleinestablishingdirectionalcuesinbilaterian generateshapeinmetazoanembryosandduringorganogenesis.The
development, include Wnt, FGF and Nodal signalling factors underlying actin behaviour has been analyzed in great detail in
(Hobmayeretal.,2000;Lengfeldetal.,2009;Philippetal.,2009; embryos of various bilaterian model organisms, and mechanisms
Lange et al., 2014; Watanabe et al., 2014). During tissue suchasapicalconstrictionandlateralintercalationhavebeenshown
recruitment, ectodermal and endodermal muscle processes finally to cause tissue bending and tissue movements. In this report, we
reinsertandelongateinproperorientationwithrespecttothebud’s have studied actin dynamics during bud evagination in the body
n
oral-aboral axis in order to re-establish proper contractility of the wall of the simple metazoan Hydra. Asexual reproduction using
e
bodycolumnofthenewanimal.Inaparallelsetofexperiments,we ‘adult’tissueisacommonfeatureamongancestralanimallineages. p
O
have studied de novo formation of muscle processes in Hydra Hence,ourfindingsmayaddtoageneralunderstandingofshape-
y
reaggregates. The results show that the establishment of parallel generating mechanisms in animal development. The body wall of g
actinfibersatthebasalsurfaceofepithelialcellsisinitiallyacell- Hydradiffersfrombilaterianembryonictissuelayersintwomajor o
l
autonomous event. Thereafter, coordinated alignment of muscle aspects: it is built as an epithelial double layerand the individual o
i
processeswithinlargerfieldsofcellsfollowstheformationofanew unit is a differentiated epitheliomuscular cell with at least two B
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