Patterned Anchorage to the Apical Extracellular Matrix Defines Tissue Shape in the Developing ...

14 Pages·2015·4.77 MB·English

by  Robert P. Ray

Article
Patterned Anchorage to the Apical Extracellular
Matrix Defines Tissue Shape in the Developing
Drosophila
Appendages of
Graphical Abstract Authors
RobertP.Ray,AlexisMatamoro-Vidal,
PauloS.Ribeiro,...,DavidHoule,
IsaacSalazar-Ciudad,
BarryJ.Thompson
Correspondence
[email protected]
In Brief
Regulationofglobaltensileforcesin
epitheliaisonemechanismof
determiningtissueshape.Rayand
Matamoro-Vidaletal.showthattissue
contraction,incombinationwithlocalized
anchoragetothecuticlebytheapical
extracellularmatrixproteinDumpy,gives
risetoanisotropictensionsthatshapethe
appendagesintheDrosophilapupa.
Highlights
d TheapicalextracellularmatrixproteinDumpy(Dp)isrequired
forappendageshape
d Dpanchorstheepidermistothecuticle,generatingtension
duringtissuecontraction
d AlterationofthepatternofDpgivesrisetopredictable
changesinappendageshape
d Narrow(Nw),Tapered(Ta),andLanceolate(Ll)affectshape
bymodulatingDp
Rayetal.,2015,DevelopmentalCell34,310–322
August10,2015ª2015TheAuthors
http://dx.doi.org/10.1016/j.devcel.2015.06.019
Developmental Cell
Article
Patterned Anchorage to the Apical
Extracellular Matrix Defines Tissue Shape
Drosophila
in the Developing Appendages of
RobertP.Ray,1,2,7,*AlexisMatamoro-Vidal,4,5,7PauloS.Ribeiro,2,3NicTapon,2DavidHoule,4IsaacSalazar-Ciudad,5,6,8
andBarryJ.Thompson2,8
1SchoolofLifeSciences,UniversityofSussex,Falmer,BrightonBN19QG,UK
2TheFrancisCrickInstitute,Lincoln’sInnFieldsLaboratory,44Lincoln’sInnFields,LondonWC2A3PX,UK
3CentreforTumourBiology,BartsCancerInstitute,QueenMaryUniversityofLondon,CharterhouseSquare,LondonEC1M6BQ,UK
4DepartmentofBiologicalScience,FloridaStateUniversity,Tallahassee,FL32306,USA
5DepartmentdeGene`ticaiMicrobiologia,Genomics,Bioinformatics,andEvolutionGroup,UniversitatAuto`nomadeBarcelona,Cerdanyola
delValle`s08193,Spain
6CenterofExcellenceinExperimentalandComputationalDevelopmentalBiology,DevelopmentalBiologyProgram,Instituteof
Biotechnology,UniversityofHelsinki,P.O.Box56,FIN-00014Helsinki,Finland
7Co-firstauthor
8Co-seniorauthor
*Correspondence:[email protected]
http://dx.doi.org/10.1016/j.devcel.2015.06.019
ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
SUMMARY mechanismsthatinfluencetissueshape:activeshapechanges
arisingfromintrinsicforcesactinglocally,whichareintegrated
How tissues acquire their characteristic shape is a overtheentiretissuetogeneratecomplexmorphogeneticmove-
fundamental unresolved question in biology. While ments,andpassiveshapechanges,whicharedrivenbyextrinsic
geneshavebeencharacterizedthatcontrollocalme- forces thatactglobally toinfluence the behaviors ofindividual
chanical forces to elongate epithelial tissues, genes cells(BlanchardandAdams,2011).
controlling global forces in epithelia have yet to be Morphogeneticprocessesdrivenbylocallyactingforceshave
been characterized in both plants and animals, and typically
identified. Here, we describe a genetic pathway that
shapes appendages in Drosophila by defining the involvedirectedcellrearrangements,orientedcelldivisions,or
acombinationofboth(LecuitandLeGoff,2007).Invertebrate
patternofglobaltensileforcesinthetissue.Intheap-
embryos, elongation of the anterior-posterior axis is driven in
pendages,shapearisesfromtensiongeneratedbycell
part by the polarized migration of mesenchymal cells that un-
constrictionandlocalizedanchorageoftheepithelium
dergoconvergentextensionmovementsundercontrolofplanar
tothecuticleviatheapicalextracellular-matrixprotein cell polarity (PCP) genes (Heisenberg et al., 2000; Tada and
Dumpy(Dp).AlteringDpexpressioninthedeveloping Smith,2000).Asimilar,PCP-dependentprocesshasbeenimpli-
wingresultsinpredictablechangesinwingshapethat catedintheelongationofkidneytubulesinXenopusandmice
can be simulated by a computational model that (Lienkamp et al., 2012). In Drosophila, anterior-posterior (A/P)
incorporates only tissue contraction and localized axiselongationalsoinvolvesconvergentextensionmovements
anchorage. Three other wing shape genes, narrow, that are driven by planar-polarized localization of Myosin II,
tapered, and lanceolate, encode components of a which constricts epithelial junctions oriented along the dorsal-
ventralaxis.Theresultingstructuresarethenresolvedintonew
pathway that modulates Dp distribution in the wing
A-P oriented junctions that drive extension of the germ band
torefinetheglobalforcepatternandthuswingshape.
(Bertet et al., 2004; Blankenship et al., 2006; Irvine and Wie-
schaus,1994;ZallenandWieschaus,2004).
Local forces can also affect tissue shape by influencing the
INTRODUCTION orientationofcelldivisions.Thismechanismisbestcharacter-
ized in the Drosophila appendages, where elongation of the
Tissuemorphogenesisdependsontheprecisecontrolofbasic proximal-distal(P-D)axisisachievedbyorientatedcelldivisions
cellbehaviorssuchascelldivision,celldeath,cellshape,and intheimaginaldiscs(Baena-Lo´pezetal.,2005).P-Delongation
cell rearrangement during development (Lecuit and Le Goff, inthediscsresultsfromtheplanar-polarizedlocalizationofthe
2007).Whiletheregulationofcellproliferationandcelldeathin atypical Myosin, Dachs, by the Fat-Dachsous planar polarity
determining tissue size have been extensively studied (Edgar, system. Dachs constricts cell junctions where it is enriched,
2006; Halder and Johnson, 2011), the mechanisms underlying alteringcellshape,andthusbiasingtheorientationofthemitotic
thecontroloftissueshapeareonlyjustcomingtolight(Lecuit spindle(Maoetal.,2011).Polarizedcelldivisionshavealsobeen
and Le Goff, 2007; St Johnston and Sanson, 2011; Zallen, implicated in other developmental processes including germ
2007).Inrecentyears,evidencehasemergedfortwotypesof bandextensioninDrosophila(daSilvaandVincent,2007),shoot
310 DevelopmentalCell34,310–322,August10,2015ª2015TheAuthors
apexandpetalmorphogenesisinplants(Reddyetal.,2004;Roll- (Figures1A–1D)(Carlson,1959).RNAisilencingofdpthroughout
and-Lagan et al., 2003), and neurulation in zebrafish (Concha thewingbladerecapitulatesthetruncatephenotypewith100%
and Adams, 1998), but the molecular mechanisms underlying penetrance (Figure 1E) and the same phenotype is produced
theseexamplesremaintobedetermined. withtheDll-Gal4driver,whichisexpressedathighlevelsonly
There is also evidence for extrinsic forces acting across tis- atthemargin(Figure1F).Dll-Gal4isalsoexpressedinlegsand
suestodrivemorphogenesis.InDrosophila,A-Ptensileforces antennae,anddepletingdpinthesetissuesresultsinretraction
arisingfromconvergenceandextensionoftheunderlyingmeso- ofthedistalsegmentsofbothappendages(Figures1Gand1H),
dermhavebeenimplicatedindrivingcellshapechangesinthe indicatingthatdpplaysageneralroleindeterminingappendage
ectodermthatcontributesignificantlytothefastphaseofgerm shape.
band extension (Butler et al., 2009). Similarly, in Xenopus,
convergence and extension of deep mesenchymal cells TheDpProteinIsLocalizedtotheApicalExtracellular
generateforcesthatpullontheoverlyingepithelialcells,which, MatrixandIsRestrictedtoDistalRegionsofthePupal
asaconsequence,undergopassiveintercalation(Keller,2002). Appendages
In zebrafish, actomyosin contraction within the yolk syncytial dpencodesagigantictransmembraneproteinthatformspartof
layergeneratesanisotropictensionintheenvelopingcelllayer theapicalextracellularmatrix(aECM)andwhoseprimaryfunc-
that drives cell shape changes, cell rearrangements, and the tionistoanchorectodermalcellstotheoverlyingcuticle(Bo¨kel
orientation of cell divisions (Behrndt et al., 2012; Campinho etal.,2005;Ja(cid:1)zwin(cid:1)skaetal.,2003;Wilkinetal.,2000).Tochar-
et al., 2013). These findings highlight the potential importance acterizethedistributionofDpproteinduringappendagedevel-
ofglobaltensileforcesinanimalmorphogenesis,yethowthese opment,wehaveusedaproteintrapinsertionintoanN-terminal
forcesarecontrolledgeneticallyremainsunknown. intronofdpthatintroducesayellowfluorescentprotein(YFP)tag
Here, weexamine the genetic control of global forces using intotheextracellulardomainoftheprotein,butdoesnotaffect
the Drosophila pupal wing as a model. Previous studies have proteinfunction(dp-YFP,seeExperimentalProcedures).Inthe
shownthatP-Delongationofthewingarisesfrompassiveorien- larvalimaginaldiscs,Dpisfoundinadensemeshworkthatuni-
tationofcelldivisionsandcellrearrangementsdrivenbyglobal formlycoverstheapicalsurfaceoftheepithelium(Figure2A).In
anisotropictensionimposedbycellconstrictionintheproximal thepupalwing,however,Dpisrestricted.Attheonsetofhinge
partofthewing(Aigouyetal.,2010).Weshowthatagroupof contraction (18 hr After Puparium Formation, APF), apically
well-known Drosophila mutants that affect wing shape disrupt localized Dp is only found at the wing margin and to a lesser
components in a genetic pathway that acts to determine the extentalongthetrajectoriesoftheL3andL5veins(Figures2B
pattern of global tensile forces in the wing. Central to this and2E),whileinthepupallegandantenna,Dpisfoundatthe
pathway is the apical extracellular matrix protein Dumpy (Dp) extremedistaltipoftheappendage(Figures2Jand2K).Astis-
thatlinksthepupalwingepitheliumtotheoverlyingpupalcuticle. suecontractionproceeds,denovoexpressionofDpaccumu-
ThepatternofDplocalizationatthecrucialtimeofhingecontrac- lates throughout the tissue (Figures 2C and 2D) such that, at
tion determines the ultimate shape of the wing. Our findings 30hrAPF,itappearsasadiaphanousnetworkofenmeshedfi-
revealageneralmechanismforthecontroloftissueshapedeter- bers thatissimilar inappearance to theaECMfoundin verte-
minationthathasimportantimplicationsforunderstanding the brates(Figures2F–2I)(Jovineetal.,2002).
evolutionofshapedeterminationinanimalsystems.
DpAnchorstheWingMargintothePupalCuticleto
RESULTS DefinethePatternofGlobalForces
OurresultssuggestamechanismwherebythelocalizationofDp
ThedumpyGeneIsRequiredtoShapetheDrosophila at 18 hr APF defines the pattern of attachment to the pupal
Appendages cuticle. In support of this view, we find that in wild-type, the
Wesoughttoidentifygenesinvolvedindefiningthepatternof wing margin is attached to the overlying pupal cuticle during
tensileforcesinthepupalwing.Wereasonedthathingecontrac- theearlyphaseofhingecontraction,ashasbeenobservedpre-
tioncouldonlyresultinanisotropictensionifthewingepithelium viously for wings cultured in vitro (Turner and Adler, 1995). By
isanchoreddistallytoprovidethemechanicalresistanceneces- contrast,indpmutants,thewingisnotattached andappears
sary to give rise to the tension. Mutants that disrupt this tofloatfreelywithinthepupalcuticle(Figures3Aand3B,arrows).
anchoring should have the normal pattern of veins and inter- Furthermore,thewingshapeofdpmutantsdivergesfromwild-
veins,butshowaretractionofthewingbladetowardthehinge. typeonlyduringpupaldevelopment,asreportedbyWaddington
Suchaphenotypeisassociatedwithallelesofthedumpy(dp)lo- (1940).At18hrAPF,thesizeandshapeofdpmutantwingsare
cus. Classical genetic studies on dp mutants revealed three not substantially different from wild-type (Figures 3C and 3D).
phenotypic states for the locus: an oblique truncation of the However, over the course hinge contraction, the dp mutant
wing(‘‘o’’),pitsonthethoraxknownasvortices(‘‘v’’),andhomo- wingbladepullsawayfromthecuticle,andthetissuecontracts
zygous lethality (‘‘l’’). While the null phenotype of the locus is intoaroundedcup-shapethatprefigurestheshapeoftheadult
lethality,dpoallelesashomozygotesorincombinationwithother wing(Figures3Cand3D,bottom;cf.Figures1Aand1E).
alleles produce a continuous spectrum of wing phenotypes Tofurthertestthenotionthatfinalwingshapearisesfromthe
rangingfromamildflatteningofthedistaltipofthewing(the‘‘ob- combination of tissue contraction and patterned anchorage of
lique’’phenotype),toacollapseofthedistaltip(theeponymous the wing margin, we developed a vertex model of the pupal
‘‘dumpy’’phenotype),and,inthemostextremecase,toacom- wingepitheliumthatincorporatescontractionofthehingeregion
plete retraction of the wing blade (the ‘‘truncate’’ phenotype) with patterned attachment of the margin to a fixed cuticle
DevelopmentalCell34,310–322,August10,2015ª2015TheAuthors 311
Figure1. ThedpGeneIsRequiredtoShape
theDrosophilaWing,Leg,andAntenna
(A–F)Wingphenotypesassociatedwithwild-type
(A) or dp loss of function (B–F). The dpo alleles
produce wing phenotypes of differing severity:
oblique (B), dumpy (C), and truncate (D). The
silencingofdpbytheexpressionofaUASRNAi
transgeneintheentirewingbladewithnub-Gal4
(E)oralongthewingmarginwithDll-Gal4(F)re-
capitulatesthetruncatephenotype(E).
(G and H) The phenotypes associated with Dll-
Gal4>dpRNAiinthesecondleg(G)andantenna
(H)comparedwiththewild-type(top).Asinthe
wing,dpknockdownresultsinacontractionofthe
distalpartoftheappendage.
down of dp in the intervein between L3
andL4withdpp-Gal4resultsinaretrac-
tion of the distal tip of the wing, where
this intervein intersects with the margin
(Figures4Band4G).Asimilarretraction
of the distal tip of the wing is seen with
sal-Gal4>dp-RNAi, but the phenotype is
more severe, reflecting the broader
expressionofthedriver(Figures4Cand
4H).Notably,thisphenotypeissimilarto
the dumpy phenotype associated with
classical dp alleles (Figure 1C). RNAi
depletion of dp in the complementary
patternwithbrk-Gal4resultsinanarrow-
ingofthewingbladeconsistentwithloss
ofanchoragealongtheanteriorandpos-
terior margins (Figures 4D and 4I),
whereas silencing dp in the posterior
compartment with hh-Gal4 results in a
contraction of the posterior part of the
blade (Figures 4E and 4J). To address
whether these phenotypes can arise as
a result of alterations in the anchorage
of the tissue, we incorporated the pre-
substrate (Figures 3E and S5 and Movie S1). In the wild-type dictedpatternsofDpfromeachofourexperimentsintoourcom-
simulation, the hinge undergoes a contraction comparable to putermodel.Theresultingsimulatedwingshapesresemblethe
whatisobservedinvivo,andthebladerespondsbyelongating corresponding in vivo phenotypes (Figures 4K–4O and Movie
alongtheP-Daxisviaorientedcelldivisionsandcellrearrange- S2). These results indicate that the pattern of Dp attachment,
ments,asdescribedpreviously(Aigouyetal.,2010).Ifthereisno coupled with contraction of the tissue, can account for the
attachment,theentirebladecontracts,producingawingshape wingshapephenotypesobservedinvivo.
thatsimulatesthenub-Gal4>dp-RNAiphenotype(Figure3Fand
MovieS1).Theseresultsindicatethatthefinalshapeofthewing Narrow,Tapered,andLanceolateAffectWingShape
canarisesimplybythepatternoftensileforcesresultingfromtis- The tapered wing phenotypes we have observed with brk-
suecontractionandpatternedanchorage. Gal4>dp-RNAi and hh-Gal4>dp-RNAi are reminiscent of the
wing phenotypes produced by three other loci, narrow (nw),
AlteringtheExpressionPatternofDpPredictably tapered(ta),andlanceolate(ll),thatwerefirstidentifiedearlyin
AffectsWingShape the last century (Meyer and Edmondson, 1949; Morgan et al.,
If the localization of Dp determines tissue shape during hinge 1925).Inactivationofthesegenesproducesarangeofpheno-
contraction,thenchangingthepatternofDplocalizationinthe types that can be generalized as a narrowing and lengthening
wing should result in predictable changes in shape. To test of the wing. The phenotypes associated with nw alleles are
this, we silenced dp in defined patterns in the wing with the dosage sensitive. Dominant antimorphic alleles (e.g., nwD/+,
driversdpp-Gal4,sal-Gal4,brk-Gal4,andhh-Gal4,thusgener- nwB/+) and weak hypomorphs produce a mild tapering of the
ating novel patterns of dp anchorage (Figures 4A–4J). Knock- distalpartofthewing(Figures5Aand5B),whilerecessivealleles
312 DevelopmentalCell34,310–322,August10,2015ª2015TheAuthors
Figure2. TheDpProteinIsanaECMComponentthatIsSpecificallyLocalizedduringMorphogenesis
(A–G)ImmunolocalizationofDpinthewild-typethirdinstarwingdisc(A),pupalwingsduringhingecontraction(B–E),inpupallegs(J),andantennae(K).
(A–GandJ–K)Dp-YFPisshowningreen,actininred,andthenucleiinblue.
(A,E,andI)Insetsshowazstackofthemainimagealongtheplaneindicatedbythearrowhead.
(A)Inlarvalimaginaldiscs,Dpisexpresseduniformlythroughouttheepitheliumandislocalizedapically.
(B–E)Inthepupalwing,expressionisdynamic:at18hrAPF,Dpisonlyfoundapicallyatthewingmargin,withweakexpressionalongthetrajectoriesofL3andL5
(BandE).Overthenext10hr,Dpaccumulatesuniformlyovertheapicalsurfaceoftheepithelium,sothatby30hrAPF,theproteinappearsinadiaphanous
networkoverlyingtheactin-richapicalmembrane(C,D,andI).
(F–H)SEMimagesrevealthedevelopmentoftheaECMnetworkbetween18–30hrAPF(thescalebarrepresents5microns).At30hrAPF,theaECMissimilarin
appearancetotheaECMthathasbeendescribedinvertebratesystems.Inthelegsandantennae,Dpisalsolocalizedintheearlystagesoftissuecontraction,
withhighlevelsoftheproteindetectedattheextremetipoftheleg(J)andattheequivalentpositionintheantenna(K).(F)–(H)showSEMimagesofthepupalwing
surfaceatthestagesindicated.
giverisetothedramaticnarrowingoftheentirewingbladeafter ures1A–1Dand4).Thus,forbothdpandnwphenotypes,alter-
whichthegeneisnamed(Figure5C).Thesamerangeofpheno- ations in blade shape are associated with changes in hinge
typescanberecapitulatedbyRNAiknockdownusingnub-Gal4 shape, suggesting that the cells of these two domains feed
or Tub-Gal4 (Figures 5D and 6A) to drive hairpin constructs back on one another according to the pattern of tensile force
directed toward different exons of the nw transcript (see Fig- onthetissue(seeDiscussion).
ureS2).Allelesoftaandll,whicharehypomorphicfortheloci
(see below), produce the weaker phenotype characteristic of nwEncodesaSecretedC-typeLectinDomain
thedominantallelesofnw(Figures5Eand5F). ContainingProtein
Morphometricanalysisrevealsthatallofthesephenotypesare To investigate the function of these loci, we genetically and
associated with a simple shape warp that is, in essence, a molecularly characterized the affected genes. Using standard
stretchofthewingalongtheP-Daxis.Dependingonthegeno- deficiencymapping,nwwaslocalizedtoasmallregionincyto-
typesincluded intheanalysis,between75%–85%ofthevari- logicaldivision54A(FigureS2).RNAiofcandidategenesinthe
anceisassociatedwithasingleprinciplecomponentthatconsti- interval identified a single gene, CG43164, that produced the
tutes an inward shift of landmarks along the anterior and nwphenotypewhenknockeddownwiththewing-specificdriver
posteriormarginsandanoutwardshiftoflandmarksatthedistal nub-Gal4(Figure5D).Confirmingthisresult,twoP-elementin-
tip of the wing (Figures S1A–S1D). Significantly, the change in sertions, G18887 and KG02048, that lie in the 50 UTR of
blade shape is also associated with a contraction of the land- CG43164 (Figure S2) fail to complement nw alleles, and
marks associated with the hinge. These data suggest that the sequencing of nwB, nwD, and nwDrS5 revealed lesions in the
stretchofthewingbladeisassociatedwithfurthercontraction CG43164codingsequence,whichwerefertohereafterasnw
ofthewinghingethanwhatisobservedinwild-type.Notably, (TableS1,seealsoFigureS2).
a corresponding deformation of the hinge is observed for any The nw gene encodes a number of different mRNA species
phenotypeforwhichthepatternofDphasbeenaltered(seeFig- producedbydifferentialinitiationfromapairofnestedpromoters
DevelopmentalCell34,310–322,August10,2015ª2015TheAuthors 313
Figure 3. Dp Anchors the Wing Margin to
the Pupal Cuticle to Define the Pattern of
GlobalForcesthatShapetheWing
(AandB)Brightfieldimagesoffrontalsectionsof
wild-type(A)andnub-Gal4>dp-RNAi(B)wingsat
18hrAPF,justafterthepupalapolysis.Inthewild-
type,theanteriorandposteriormarginofthewing
blade(wb),butnotthedorsalandventralsurfaces,
are attached to the overlying pupal cuticle (pc)
(A,arrows),whileinthedp-RNAi,theepitheliumis
fullydetached(B,arrows).Consequently,phalloi-
dinstaining(red)revealsthatthewild-typewing
remainsapposedtothecuticleduringtheperiod
from18–24hrAPF(C),while thedp-RNAiwing
retractsproximally(D).
(CandD)Thepositionofthecuticleisindicatedin
thefinalpanelbyadashedwhiteline.
(E and F) Epithelial vertex model of pupal wing
morphogenesis.Thestartingpointofthesimula-
tionistheearlypupalwingshape,withthehinge
regionshowninlightblueandthebladeinred.The
wingveinsareshownindarkblue.Thecontraction
ofthetissue,moststronglyinthehinge region,
combined with anchorage of the wing margin
(green lines) are sufficient to simulate wild-type
wingmorphogenesis.Whentheanchorageofthe
marginisabsentinthecomputermodel,theentire
wingretracts,simulatingthedpmutantwing(see
MovieS1).
C-TypeLectinDomain(CTLD).Thecore
CTLDconsistsoffourCysteine(Cys)res-
idues that are laid out with respect to a
characteristic arrangement of a helices
andbsheets.TheseCysresiduesformin-
tramolecular disulfide bridges that give
risetothedouble-loopfoldcharacteristic
of the domain (Zelensky and Gready,
2003) (Figure 5G). Additional Cys resi-
duesmayalsobepresentand,generally,
ifthenumberiseven,allformintramolec-
ular bonds, while if the number is odd,
oneoftheCyswillformanintermolecular
bondeitherwithanotherCTLDproteinor
an unrelated protein (Drickamer and
Dodd, 1999). The Nw CTLD contains
five Cys residues, implying that one will
form an intermolecular disulfide bridge,
and given the placement of these resi-
dueswithrespecttothesecondarystruc-
ture, the final Cys is most likely to be
involvedinthisbond(seeFigure5G).
WefindthatNw-SandNw-Laresolu-
ble,secretedproteinsandeitherisoform
canbeimmunoprecipitatedfromthecul-
ture medium when the nw gene is ex-
pressedinDrosophilaS2cells(Figure5H).
andbydifferentialsplicingandterminationofthefinalexon.The Undernon-reducingconditions,theproteinsrunpredominantly
varioustranscriptsencodetwoproteinisoforms,whichwerefer as higher molecular weight complexes that are approximately
toasNw-Short(Nw-S)andNw-Long(Nw-L)(FigureS2).Thetwo twice the size of the respective monomers, suggesting that
isoformsshareacommonN-terminalmotifwithhomologytothe they can form dimers. Additionally, when Nw-S and Nw-L are
314 DevelopmentalCell34,310–322,August10,2015ª2015TheAuthors
Figure4. AlteringthePatternofDpGivesRisetoPredictableChangesinWingShape
(A–E)Adultwingphenotypesforwild-type(A),localizedsilencingofDpwithdpp-Gal4(B),sal-Gal4(C),brk-Gal4(D),hh-Gal4(E)(top),diagramsshowingthe
regionofthewingwhereDpissilenced(green),andthecorrespondingpatternofDpanchorage(redasterisks)(bottom).
(F–J)Developmentaltimecourseofpupalwingdevelopmentrevealedbyphalloidinstaining(red)showingtheontogenyoftheshapechangefrom18to24hrAPF.
Thepositionofthepupalcuticlewithrespecttothewingisshowninthefinalpanel(dashedwhiteline).
(K–O)Thefinalimagesfromthecomputationalsimulationsofthegenotypescorrespondingto(A)–(E),usingtheepithelialvertexmodel(seeMovieS2).Thewing
bladecellsareshadedred,thehingecellsareshadedlightblue,andtheveincellsareshadeddarkblue.Thedashedwhitelinesmarkthewingoutlineatthe
beginningofthesimulation.TheDpanchoragetothecuticleisshownasgreenlinesendingwithgreendotsandtheanchorageinthehingeasgreendots.
co-expressed, they form complexes corresponding to all Monooxygenase(PAM).Furthercharacterizationofthetwoloci
possiblehomodimericandheterodimericcombinations.Thefor- confirmedthisidentification.Thetwollallelesfailtocomplement
mation of these higher molecular weight complexes is depen- Phmk07623(Figure5E),aP-elementinsertionintothePhmopen
dent on the presence of the final Cys, as when this residue is reading frame, and they are associated with the same 10 bp
mutated only the monomeric form is observed (Figure 5H). deletionattheCterminusofthePhmproteinthatremovesthat
Finally, when FLAG-tagged Nw-S is co-expressed with HA- final26aminoacids(aa)andappends79aaor80aafromthe
taggedNw-SorNw-L,immunoprecipitationwithanti-FLAGan- secondframe(FigureS3;TableS1).Theta1alleleisassociated
tibodies co-precipitates the HA-tagged proteins (Figure 5H). withanonsensemutationinthesecondcodingexonofPal1(Fig-
Furthermore,ifthefinalCysismutated(Cys>Ala),themodified ureS3),andRNAiofPal1inthedevelopingwingphenocopies
proteinisexpressed,butcannotdimerizeeitherwithitselforthe thetamutantphenotype(Figure5F;TableS1).
wild-typeprotein(Figure5H). The a-amidation catalyzed byPAM isspecific for a terminal
glycineresidue(Eipperetal.,1992),andconsistentwithNwbe-
taandllAreRequiredtoActivatenw ing a target for this modification, the short form of Nw (Nw-S)
Giventhesimilaritybetweenthemutantphenotypesof nw,ta, ends with a glycine residue that is universally conserved in all
andll,itwaslikelythatthegenesarepartofacommonbiochem- Nworthologs.Furthermore,themodificationistypicallyassoci-
ical pathway. Initial characterization of ta and ll by deficiency ated with small proteins, such as neuropeptides, whose func-
mapping, localized the loci to small intervals containing eight tionmaybedisruptedbytheionizationoftheterminalcarboxyl
andninegenes,respectively(FigureS3).Consideredseparately, group(Eipperetal.,1992).ThefactthatNw-Sisasmallprotein
neitherintervalcontainedanobviouscandidateforaproteinthat (194 aa), that is predicted to make a tight fold (Zelensky and
mightinteractwithNw.Consideredtogether,however,twocan- Gready, 2005), makes it a likely target for a-amidation, and
didateswereimmediatelyapparent:thetaintervalincludesthe the mutant phenotypes of ta and ll indicate that thismodifica-
Drosophila ortholog of Peptidyl-a-hydroxyglycine a-Amidating tionisessentialforNwfunction.Takentogether,ourdatasug-
Lyase(Pal1),andthellintervalincludestheorthologofPeptidyl- gestthatNw,Ta,andLlworkin acommonpathway,withthe
glycinea-HydroxylatingMonooxygenase(Phm),thetwocompo- modifying enzymes being required for the maturation of Nw
nents of the widely conserved Peptidylglycine a-Amidating (Figure5I).
DevelopmentalCell34,310–322,August10,2015ª2015TheAuthors 315
(legendonnextpage)
316 DevelopmentalCell34,310–322,August10,2015ª2015TheAuthors
Figure 6. Localization of Nw in the Devel-
oping Wing and the Ontogeny of the Nw
Phenotype
(A–E)LocalizationofNw-GFPthathasbeenex-
pressedundercontrolofthenub-Gal4driver.The
Nw-GFPisshowningreen,actininred,andthe
nucleiinblue.Throughoutwingdevelopment,Nw
followsthepatternofDplocalization:inthewing
disc,itisfoundapicallythroughouttheepithelium,
butat18hrAPF,itislocalizedtothewingmargin
and the trajectories of L3 and L5 (B), as wing
contractionproceeds,Nwaccumulatesuniformly
throughout the wing blade in a diaphanous
networkoverlyingtheactin-richapicalmembrane
andbristles(C–E;seealsoFigureS4).
(F–I)(F)Adultwingshowingthephenotypeasso-
ciated with Tub-Gal4>nw-RNAi (A), which is
similartothatproducedbystronglossoffunction
allelesofnw(seeFigure5C).Likethedpmutant
phenotype(seeFigure3),theshapedefectasso-
ciatedwithnwmutantsarisesbetween18–24hr
APF,concomitantwithhingecontraction(G–I;cf.
Figures3Cand3D).
(J) Computational simulation of the nw mutant
phenotypeusingtheepithelialvertexmodel.
duringhingecontraction(Figures6A–6D),
aswehaveobservedforthedpmutations
(cf. Figures 3C and 3D). In addition, the
expressionofaNw-GFPfusiondrivenby
the nub-Gal4 driver precisely follows the
patternofDpexpressionthroughoutwing
development,despitethefactthatfusion
protein is expressed in all cells of the
wingblade.Inthewingdisc,Nwislocal-
ized apically, in the pupal wing at 18 hr
APF,itislocalizedtothewingmarginand
subsequentlyitaccumulatesuniformlyin
a fibrous network over the entire epithe-
lium(Figures 6F–6J).Moreover, Nw-GFP
doesnotlocalizetotheaECMindpmutant
nwAffectstheLocalizationofDptoControlWing wings,inwhichtheaECMfailstoformproperly(FigureS4).Finally,
Morphogenesis nwanddpinteractgenetically,withtransheterozygotesproducing
Themolecularcharacterizationofnw,ta,andll,andthemutant wingsthatareeitheracutelytaperedorretractedtowardthehinge,
phenotypetheyproduce,suggestthattheymightbeinvolvedin similartodpmutants(Figures7A–7G).Theseresultssuggestthat
definingthelocalizationofDpinthedevelopingwing.Consistent nw mutants affect wing shape by influencing the pattern of
withthisidea,theshapedefectassociatedwithnwmutantsarises anchorageofthewingepitheliumtotheoverlyingcuticle.
Figure5. Thenw,ta,andllGenesControlWingShape
(A–F)Adultwingsshowingshapephenotypesassociatedwithnw,ta,andllmutants.Comparedtowild-type(A),thenwDheterozygote(nwD/+)ismildlytapered
distally(B),whiletherecessivehemizygote(nw2/Df)isnarrowerandlonger(C),similartowhatisobservedwithnub-Gal4>nw-RNAi(D).Thesephenotypesare
associatedwithasingleshapewarpconsistingofastretchofthewingbladealongtheP-Daxis(seeFigureS1).Thelossoffunctionofll(orPhm)ortabyRNAi
knockdownresultsinamildnarrowingofthewing,similartothephenotypeproducedbynwD/+(EandF).
(G)MolecularcharacterizationofnwrevealedthatitencodesaCTLDprotein(seealsoFigureS2),andsecondarystructureoftheNw-Sproteincomparedtothe
canonicalCTLDstructureshowsthetwodisulfidebridges(reddottedlines)thatstabilizethecharacteristicfoldofthemotif,plusanadditionalCysattheC
terminus(bluedottedline),whichisinvolvedindimerization.
(H)BiochemicalanalysisofNwshowsthatbothproteinisoformsaresecretedintothemediumwhenexpressedintissueculturecells(left)andthatundernon-
reducingconditionstheyarepredominatelyfoundashomo-orhetero-dimers(right).Dimerization,butnotsynthesisorsecretion,dependsonthefinalCysshown
in(G):mutationofthisCysblocksboththeformationofdimersandtheabilityofthemutantformtocoIPthewild-typemonomer(right).
(I)Molecularcharacterizationoftaandll(seeFigureS3)revealsthattheyencodethetwoenzymes,Pal1andPhm,thatcatalyzea-amidation,apost-translational
modificationthatconvertsaC-terminalglycineresidueintoana-amide.Nw-Sterminateswithaglycineand,giventhephenotypesoftaandll,a-amidationofthis
residuemustbeessentialforNwfunctioninvivo.
DevelopmentalCell34,310–322,August10,2015ª2015TheAuthors 317
ToinvestigatewhetherNwhasaneffectonDp,weexamined
the distribution of Dp-YFP in nw mutant wings. Given the nw
phenotype,andtheresultsfromoursimulations,wewouldpre-
dictthatDpislocalizedtotheverydistaltipofthewing.Indeed,
innwmutantwingsat18hrAPF,Dp-YFPisrestrictedtoanarrow
crescentattheextremedistaltipofthewingandisabsentfrom
therest of thewing margin (Figures7Hand 7I).Moreover,the
shapeofthenwmutantwingcanbesimulatedinourepithelial
vertex model simply by restricting the anchorage of the wing
margintothedistaltip(Figure6E).Together,ourdatasupport
amodelwherenwaffectswingshapebyalteringtheprofileof
the Dp localization at the onset of hinge contraction. The nw,
ta,ll,and dpgenesthusacttogetherto definewing shapeby
controlling the pattern of global forces acting across the wing
duringpupalmorphogenesis.
DISCUSSION
AGeneticMechanismthatDefinesTissueShape
Here,wehaveidentifiedagroupofgenesthatdefinetheglobal
forcepatternsthatshapetheappendagesinDrosophila.During
pupaldevelopment,shapeisdeterminedbyageneralcontrac-
tion of the tissue in combination with localized anchorage to
the pupal cuticle, which is mediated by the aECM protein Dp.
Inthedevelopingwing,Dpislocalizedtothewingmarginsuch
that, as tissue contraction proceeds, tension along the P-D
axiselongatesthewingandalsodrawsthetwowingsurfaces
together.Indeed,manipulatingthepatternofDplocalizationat
thisstageleadstodramaticchangesinwingshapethatreflect
the underlying change in tissue anchorage. In the legs and
antennae,Dpisfoundinadenseplaqueatthedistaltipofthe
appendage, and, as in the wing, tissue contraction results in
taperingandelongationofthestructure.Thus,wehaveidentified
a genetic mechanism that determines shape by regulating the
patternofglobaltensileforcesthattheepitheliumexperiences
duringtissuecontraction.
EstablishingthePatternofDpLocalization
Whilethemechanismwehaveuncoveredisclearlyimportantfor
properanchorageofthewingepitheliumtothecuticle,itisonly
onepartoftheregulatorymechanismthatleadstothelocalized
attachment.Indeed,inanwmutant,whilethedistributionofDp
isaltered,itisstilllocalizedtothemargin,thusotherinputsmust
beinvolvedindefiningwhereDpislocalizedduringpupaldevel-
opment.Inthewing,thelocalizationofDptothemarginisremi-
niscentoftheexpressionofgenescontrolledbytheNotchand
Figure7. GeneticandMolecularInteractionsbetweennwanddp Winglesspathwaysthatdefinethedorsal-ventralcompartment
(A–F)Thedosagesensitivityofnwmakesitidealfortestinggeneticinteractions boundary.Indeed, the geneDllisadownstream target ofWg,
withotheraECMproteins.nwshowsastronginteractionwithdp,butnotwith
andknockingdowndpinthecellsthatexpressDllphenocopies
otheraECMcomponents(datanotshown).Adpnullheterozygote(dpolvR/+)
the dp loss-of-function phenotype (Figure 1F). Moreover, the
producesawild-typewing(A),whilethenwD(ornwB)heterozygoteexhibitsa
mildlytaperedwing(B).Inthetransheterozygouscombination,dpolvR+/+nwD,a notchingassociatedwithmutationsintheNotchandWgpath-
spectrumofphenotypesisproduced,rangingfromsharplytaperedtoacomplete ways,aswellastheirtargetssuchascut,are,inessence,de-
retractionofthewingbladeresemblingthedplossoffunctionphenotype(C–F). fectsintheanchorageofthemarginduringpupaldevelopment:
(G) Distinct phenotypic classes from this spectrum of phenotypes were thefailuretospecifythemarginresultsinagapintheexpression
defined to quantitate the enhancement, shown as percent of total wings of Dp which produces a phenotype not unlike that which we
showingthephenotype.
observeindpp-Gal4>dpRNAi(Figure4B).Consistentwiththis,
(HandI)LocalizationofDpproteininwild-type(H)andnwmutantwings(I).In
ithaspreviouslybeenshownthatthenotchingassociatedwith
thewild-type,Dpproteinisdetectablethroughoutthewingmargin,withhigher
levelsatthedistaltip(H).Inthenwmutantwings,theexpressionisreducedto cutarisesduringpupaldevelopmentduringtheperiodofhinge
asmallcrescentofexpressionatthedistaltip(I). contraction (Jack et al., 1991). Thus, it may be that these
318 DevelopmentalCell34,310–322,August10,2015ª2015TheAuthors