The Hox genes Ultrabithorax and abdominal-A specify three different types of abdominal ...

14 Pages·2014·4.63 MB·English

The Hox genes Ultrabithorax and abdominal-A
specify three different types of abdominal
appendage in the springtail Orchesella cincta
(Collembola)
Konopova and Akam
KonopovaandAkamEvoDevo2014,5:2
http://www.evodevojournal.com/content/5/1/2
KonopovaandAkamEvoDevo2014,5:2
http://www.evodevojournal.com/content/5/1/2
RESEARCH Open Access
The Hox genes Ultrabithorax and abdominal-A
specify three different types of abdominal
appendage in the springtail Orchesella cincta
(Collembola)
Barbora Konopova* and Michael Akam
Abstract
Background: InDrosophilaand many other insects, theHox genes Ultrabithorax (Ubx) and abdominal-A (abd-A)
suppress limb formation onmost or allsegmentsof the abdomen. However, a number of basal hexapodlineages
retain multiple appendagesontheabdomen. In thecollembolans or springtails, three abdominal segments
develop specialized organs that originate from paired appendage primordia which fuseat themidline: thefirst
abdominalsegmentbearsthecollophore(ventraltube),involvedinosmoregulation;thefourthsegmentbearsthe
furca,theleapingorgan,andthethirdsegmentbearstheretinaculum,whichretainsthefurcaatrest.Ubxandabd-A
areknowntobeexpressedinthespringtailabdomen,butwhatroletheyplayinspecifyingthesedistinctabdominal
appendagesisnotknown.Thisislargelybecausenogeneticmodelhasbeenestablishedincollembolansoranyother
non-insecthexapod.
Results:WehavedevelopedaconvenientmethodforlaboratorycultureofthecollembolanOrchesellacinctaon
definedmedia,amethodforin-situhybridizationtoembryosandaprocedureforgeneknockdownbyparental
injection of double-stranded RNA (RNAi). We show that Orchesella Ubx transcripts are detectable in the first to
thirdabdominalsegments,andabd-Atranscriptsinthesecondtofourthsegments.KnockdownofOc-Ubxleadstothe
homeotictransformationofthecollophoreintoapairofwalkinglegs(amoreanterioridentity)buttheretinaculuminto
afurca(amoreposterioridentity).KnockdownofOc-abd-Aleadstothetransformationoftheretinaculumintoacollophore
andofthefurcaintolegs(bothanteriortransformations).SimultaneoussilencingofbothOc-UbxandOc-abd-Atransformed
allthreeoftheseappendagesintopairedlegs,butdidnotcauseappendagestodeveloponthesecond,oronthemost
posteriorabdominalsegments.
Conclusions:Weconcludethat,inOrchesella,Oc-UbxalonespecifiesthecollophoreonthefirstandOc-abd-Aalone
specifiesthefurcaonthefourthabdominalsegment.Oc-UbxandOc-abd-Afunctiontogether,apparentlycombinatorially,
tospecifytheretinaculumonthethirdsegment.TheefficiencyofRNAiinOrchesellamakesthisanattractivemodelfor
furthergeneticstudiesofdevelopmentandphysiologyinbasalhexapods.
Keywords:abdominal-A,Appendagespecification,Collembola,Evolution,Hox,Homeosis,RNAinterference,Segment
identity,Ultrabithorax
*Correspondence:[email protected]
LaboratoryforDevelopmentandEvolution,DepartmentofZoology,
UniversityofCambridge,DowningStreet,CambridgeCB23EJ,UK
©2014KonopovaandAkam;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe
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distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.TheCreativeCommonsPublic
DomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthis
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Background identity [5,6]. These appendages arise as paired bilateral
Springtails(Collembola),ProturaandDiplura,togetherwith buds on A1, A3 and A4, but in late embryos each pair
the insects proper, form a clade of arthropods called the fuses at the ventral midline to form a specialized organ:
Hexapoda.Adefiningfeatureofhexapodsisthattheirbody the collophore (also called the ventral tube) on A1 par-
has six legs, a pair growing from each of the three thoracic ticipates in water absorption [7-9]. It is linked by a
segments[1,2].Inlarvaeandadultsofthemorebasalhexa- groove in the cuticle (the ventral groove, linea ventralis)
pod lineages, such as the springtails, Protura, Diplura and to the labial glands, which probably function as excretory
wingless insects, small limbs or other appendages also de- organs; together, these organs may function as a bipartite
velop on the abdomen. In this respect, these basal lineages ‘kidney’ reducing water loss through excretion [10,11].
resemblemanycrustaceans,fromsomelineageofwhich The retinaculum on A3 functions as a clip for the furca,
the Hexapoda originated [2-4]. In the winged insects which is a powerful structure formed by the appendages
(Pterygota),abdominal appendagesmaydevelop inlarvae, onA4 that is used for jumping(Figure 1). Based on mor-
butarelostduringmetamorphosistotheadult. phological and embryological observations, it has been
The abdomen of springtails comprises six segments. proposed that the fused proximal parts of the abdominal
Three of these bear appendages, each with a unique appendagesarehomologouswiththecoxopoditesandthe
Figure1Oc-Ubx(RNAi)andOc-abd-A(RNAi)leadtohomeotictransformationofOrchesellaabdominalappendages.Allpanelsshowfirst
instarOrchesellalarvae,whosemotherswereinjectedwithdsRNAs.(A-C)lateralviews,headontheleft;(D-F)ventralviews,anteriortothetop.
(A,D)ControlRNAi.Thelarvaelooknormal.Theyhavethreepairsofthoraciclegs,whichareallvisiblein(A),acollophoreonA1,aretinaculum
onA3,andafurcaonA4;theretinaculumishiddenin(A),butvisibleandenlargedintheinsetin(D).Inboth(A)and(D)thecollophoreisfalse
colouredinmagenta,theretinaculuminyellowandthefurcaingreen.(B,E)Oc-Ubx(RNAi)larvaeareabnormalinthatthecollophoreisreplaced
byapairofwalkinglegsonA1andtheretinaculumisreplacedbyafurcaonA3;thehomeoticfurcaishiddenin(B),butvisiblein(E).(C,F)
Oc-abd-A(RNAi)larvaeareabnormalinthattheyhaveacollophoreonA3andlegsonA4.Thehomeoticallytransformedappendagesarelabeled
inyellowinallpictures.T3,A1,A3andA4in(A-C)markthethirdthoracic,thefirst,thethirdandthefourthabdominalsegments,respectively;in
(D-F)theirappendages.Thewhitearrowheadin(E)pointstotheventralgroove.c,collophore;f,furca;r,retinaculum;wl,walkingleg.Scalebars:
in(A),200μmfor(A-C);in(D),200μmfor(D-F);inset,10μm.
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distalparts,whichdonotfuse,arehomologoustothetel- embryos are either inaccessible or difficult to work with
opoditesofotherarthropodappendages[6]. [40,41]. The twin aims of our research were therefore: (1)
In general, the distinct identities of segments in insects to find a springtail that would be amenable for develop-
are specified by Hox genes [12]: in the absence of Hox mentalandfunctionalgeneticexperiments,and(2)tofind
gene function, all segments of the trunk develop similarly out how Ubx and abd-A specify collembolan abdominal
(reviewed in [13]). The Hox genes encode transcription appendages.
factors that modulate many aspects of segment develop- HerewepresentourstudyonUbxand abd-Afunction
ment, by interacting with a large number of downstream by parental RNAi in the springtail Orchesella cincta
targets [14]. They are expressed from head to tail in par- (Entomobryomorpha). Orchesella is a surface-dwelling
tiallyoverlapping domains, buttypically,themoreposter- soilspringtailthatreachesabout4mminsize.Ithassexual
iorly expressed gene dominates phenotypically over the reproduction. It has previously been used for ecotoxico-
more anterior one. Changes in the domains of expression logicalexperiments[42],anditsgenomehasrecentlybeen
ofthesegenes,andchangesinthesetofdownstreamtargets sequenced(D.Roelofs,personalcommunication).Wehave
thattheyregulate,havebeenshowntoplayasignificantrole generatedanembryonictranscriptome.Weshowherethat
intheevolutionofarthropoddiversity[15-24]. Orchesella can be raised on cultured algae and yeast as a
Ininsects,thedistinctidentitiesofdifferent segmentsin foodsourceandthattheembryosareaccessibleforin-situ
the pre-genital abdomen are specified by the Hox genes hybridization, albeit with some difficulty. We also show
Ultrabithorax (Ubx) and abdominal-A (abd-A) (reviewed that parental RNAi is simpleand effectiveforgeneknock-
in[25,26]),andbyAbdominal-B,which playsamajorrole down in this species. Using this technique, we show that
in the genital segments, and a minor role more anteriorly both Oc-Ubx and Oc-abd-A have unique limb modulatory
[27-29].WedonotconsiderAbd-Bfurtherhere. functions:Oc-Ubxonitsownspecifiesthecollophore,while
InDrosophila,andmostotherinsectsstudied,Ubxisini- Oc-abd-A on its own specifies the furca. Both Oc-Ubx and
tiallyexpressedwithinA1(strictlyspeaking,fromthepara- Oc-abd-Ajointlyspecifytheretinaculum.
segment 5/6 boundary back) while abd-A is expressed
fromA2(parasegment6/7boundary)back[26,30-33].Itis Methods
relatively well understood how this early Ubx and abd-A Orchesellacinctaculture
expression represses limbs in Drosophila [34,35]. Slightly AcultureofthespringtailofOrchesellacinctawasobtained
later in development, Ubx expression extends anteriorly fromtheVrije UniversiteitAmsterdam (kindlyprovidedby
into the thorax. This later expression is unable to repress Nico van Straalen, Dick Roelofs and Janine Mariën).
limbdevelopment,butitmodifiestheidentityofthelimbs Springtails were kept at 25°C with a photoperiod of
thatform[22,35,36]. 18 h light and 6 h dark in Petri dishes on a solid base
While both Ubx and abd-A suppress appendages in made from plaster of Paris mixed with charcoal, cov-
Drosophila, in the beetleTribolium, and probably also in ered with a layer of the alga Pleurococcus sp. (obtained
many other less derived insects, Ubx does not repress fromCCAP:theCultureCollectionofAlgaeandProtozoa,
appendage development in A1. Instead, it specifies on Argyll, Scotland, UK) and a little baker’s yeast to serve
A1 the development of a pair of glandular appendages as food. The cultures were moistened with algal liquid
called pleuropods, which mature in the embryo and are culture twice a week. Algae were grown in conical
shed at or before hatching [37,38]. Knockdown of Ubx flasks with 3 N-BBM+V medium (recipe from CCAP
in embryos results in homeotic transformation of the website,http://www.ccap.ac.uk)standingonawindow-
pleuropodstowardsthephenotype ofawalkingleg. sill. To prepare the dishes for springtails, the grown
How Ubx and abd-A might function to specify the mul- algal culture was poured onto Petri dishes with a solid
tiple distinct abdominal appendages of basal hexapods is base. Algae were left to settle for a few days, after
not known. An antibody that detects both Ubx and Abd-A which time, the liquid medium was poured out and
wasusedbyPalopoliandPatel[39]toshowthateitherone soakedupwithapieceoftissue.
orbothoftheseproteinsispresentineachoftheabdominal
appendagesofthespringtailsFolsomiaandXenylla,consist- Genecloning
entwiththehypothesisthattheseproteinsplaysomerolein Genes were isolated from cDNA prepared from mixed
appendage specification, but this reagent cannot discrimin- stages of embryos by using the ThermoScript reverse
atebetweenthetwoHoxproteins. transcriptase (Invitrogen, Carlsbad, CA). The fragment
Despite their phylogenetic position at the base of the of Oc-Ubx used for RNAi was obtained by semi-nested
Hexapoda, no springtail species has been established for PCR with the Ex Taq DNA polymerase (Takara, Japan)
wide use in comparative developmental genetics, perhaps and degenerate primers. All other isolations were done
because most species are very small, the development of withthePhusionDNApolymerase(Finnzymes,Finland).
the larger species is often slow, and in many cases their Gene-specific primers were designed on the basis of
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sequences recovered in an embryonic transcriptome microscopewithLeicaDFC300FXcameraandaLeicaTCS
(Specialist Sequencing and Bioinformatics Services SP5 confocal microscope. For scanning electron micros-
were provided by the EASIH, University of Cambridge). copy,larvaewerefixedin80%ethanol,post-fixedwithos-
The primers are listed in (Table 1). The fragments were mium tetroxide, dehydrated through an ethanol series,
cloned into the pCR 4Blunt-TOPO vector (Invitrogen, critical point dried, gold coated and observed on a FEI/
Carlsbad, CA). The sequences were aligned in the CLC Philips XL30 FEGSEM microscope. The photos were
Sequence Viewer 6 program. Possible phosphorylation adjusted using Adobe Photoshop (version CS5) and Fiji.
sites were identified using the NetPhos Server (http://
www.cbs.dtu.dk/services/NetPhos/ and http://www.cbs.
Results
dtu.dk/services/NetPhosK/).
LaboratorycultureofOrchesellacincta
InAmsterdam,thesourcecultureofOrchesellawasmain-
In-situhybridization
tained on twigs from trees overgrown by algae and steril-
Femaleslayeggsindividuallyonthesurfaceofthe culture
ized byfreezing (J.Mariën, personal communication). The
media. Eggs were collected from the Petri dishes using a
algae and fungi on the twigs are a vital source of food
fine soft paintbrush, fixed overnight in 4% formaldehyde
[45,46]. Because this method does not enable quick and
inPBS+0.1%Tween20andstoredinmethanolat−20°C.
easy inspection of the culture and timed egg collections,
Eggshells (the blastodermic cuticles) [43] were manually
we developed a method of culturing on Petri dishes with
removedusingforceps.Sampleswereprocessedaccording
definedmedia.InourculturingmethodwefeedOrchesella
to a published protocol [44] with the modification that
thealgaPleurococcus(syn.Desmococcus),becausethisalga
PBS+0.1% Tween 20 was used for washes and
waspreviouslyfoundtobethatmostconsumedbyOrches-
hybridization was at 55°C. The probe lengths were 1,251
ella in its natural habitat. Baker’s yeast forms the fungal
basesforOc-Ubxand1,230basesforOc-abd-A.
component in the diet. All individuals in the dishes can
easily be inspected, anesthetized by filling the dish with
ParentalRNAi
CO ,andtappedintoafreshdish.
dsRNAsatlengthsof725basesforOc-Ubxand1,230bases 2
forOc-abd-AweresynthesizedusingtheT3andT7Mega-
script kits (Ambion, Austin, TX). Orchesella females were SequencesoftheOrchesellaUbxandabd-Agenes
anesthetized on a carbon dioxide plate and injected with No full-length Hox protein sequences have previously
dsRNAs at a concentration of 5 μg/μl until the abdomen been reported for springtails, though short Hox frag-
was obviously inflated. After an overnight recovery, the ments have previously been isolated from the springtail
females were kept together with males of a similar age. Folsomia candida. We isolated cDNAs for the full
To check that the RNAi lowered levels of the targeted protein-coding regions of Ubx and abd-A from embryos
mRNAs,weperformedin-situhybridizationontheOc-Ubx of Orchesella. Both genes contain the expected con-
(RNAi)andOc-abd-A(RNAi)embryosandcomparedthein- servedsequencesignatures,whichincludethehomeodo-
tensity of staining with that in control embryos (Additional main (HD), hexapeptide motif (HX) and UbdA peptide
file 1). We detected a substantial reduction in staining (Additional file 2 and Additional file 3). Two isoforms
intensity. were recovered for both Oc-Ubx and Oc-abd-A. The iso-
forms differ in the length of the linker region (LR) be-
Microscopy tween the hexapeptide and the homeodomain (8 vs 12
Samples for expression analyses were observed on a Zeiss amino acids in Oc-Ubx; 27 vs 49 in Oc-abd-A). The
Axioskop2 MOT plus compound microscope with a Zeiss linkerinthelongisoformofOc-abd-Aisnoticeablylong
AxioCam MRm camera, a Zeiss Axiophot compound comparedwiththatinotherarthropodabd-Asequences.
Table1Primersforcloning
Fragment Primers(5′-3′)
Forward Reverse
Oc-UbxRNAi ATGAAYTCBTAYTTYGANCA TTCATICKICKRTTYTGRAACCA
ATYTTDATYTGICKYTCIGTIARRCAa
Oc-Ubxprobe ACATGTATGTATTTTTCACCCTT AGATGTATATTTTAGGTAAAGTTAC
Oc-Ubxfull-lengthprotein TCAATGAACTAATTTAGGAACAATAC AGATGTATATTTTAGGTAAAGTTAC
Oc-abd-ARNAiandprobe TTGGCAATCAGTGTGGTCAGG ATATACCACACCAAGCCCACA
Oc-abd-Afull-lengthprotein TTAGGGAATACAGAGGGTTCT ATATACCACACCAAGCCCACA
aNestedprimerinthesecondPCR.
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A common feature of insect Ubx sequences is the next most anterior segment (that is,T3p for Oc-Ubx; A1p
presence of two motifs C-terminal to the UbdA peptide; forOc-abd-A),astheydoinmanyinsects.
the QAQA motif and the poly-alanine stretch, which
were both shown to be important for the limb repressive
role of Ubx in insects [47,48]. The QAQA motif, but not KnockdownofOc-Ubxleadstohomeotictransformation
the poly-alanine stretch, was found in Folsomia Ubx. oftheA1andA3appendages
Similarly, Oc-Ubx contains the QAQA motif, but lacks To understand the function of Oc-Ubx and Oc-abd-A in
the poly-alanine stretch suggesting that this is a com- Orchesella, we reduced the levels of gene function in em-
mon feature of springtail Ubx. The C-termini of Ubx bryosbyinjectingdouble-strandedRNAforeachgeneinto
from diverse arthropods, but not the insects, contain the abdomens of female parents (a process colloquially
several phosphorylation sites; the phosphorylation at known as ‘knock down by parental RNAi’) [50]. In
these sites was shown to block the general ability of Ubx Tribolium, and an increasing number of other insect
to repress appendages [48]. The C-terminus of Folsomia species tested, this has been shown to reduce gene
Ubxcontainsonephosphorylationsite.Wedidnotfindany function, though there is considerable variability in both
predicted phosphorylation sites in Oc-Ubx C-terminal to thedegreeanddurationofknockdownfordifferentgenes,
theUbdApeptide,suggestingthatthesinglesiteinFolsomia andindifferentspecies[51,52].
Ubx is not a common feature of springtail Ubx. The C- As a first step, we injected two types of control dsRNA
terminioftheinsectabd-Asequencesaretypicallyenriched targeted against sequences that were not expected to be
in the amino acid glutamine, but we did not find any glu- present in Orchesella; the egfp gene of jellyfish and the
tamine residues in the C-terminus of Oc-abd-A. The TD MalE gene of E. coli. Neither of these dsRNAs interfered
motif, which is a site for interaction with cofactors, and is with normal development of the females or their progeny
present in insect but not other arthropod abd-A sequences (Table2,Figure1A,D).
[49],ismissinginOc-abd-A. Larvae from the females injected by Oc-Ubx dsRNA also
Insummary,theOc-Ubxsequenceshowsacombination hatchednormally,but76.5%ofthelarvaethathatchedfrom
ofancestralandnovel(insect)features,whiletheOc-abd-A eggs collected within the first 10 days after the injections
is more similar to abd-A sequences from arthropods other died at the end of the first instar and showed homeotic
thaninsects. transformationoftheA1andA3appendages(Table2).The
collophore on A1 was transformed into a pair of walking
legsandtheretinaculumonA3intoastructureresembling
EmbryonicexpressionofOc-UbxandOc-abd-A thefurca(Figure1B,E).
To distinguish between the expression domains of Oc-Ubx By external observation, the ectopic pair of legs on A1
andOc-abd-AinOrchesellaembryos,westudiedthedistri- was,inthemoststronglytransformedindividuals,identical
butionoftheirtranscriptsbyin-situhybridization.Springtail with the thoracic legs: the proximal parts of the A1 legs
embryosarenoteasytohandlebecausetheyaresmall,they were separate (Figure 1E), unlike the fused appendages of
aretightlybentduringthecriticaldevelopmentalstagesand the normal collophore, and the appendages ended with a
theyaresurroundedbyhighlyrefractileyolk.Wecombined claw(Figure3B;comparewithFigure3A).Thelegsmoved
NBT/BCIPstainingandbrightfieldmicroscopy,whichpro- when the larvae walked (Additional file 4). The ventral
vides low background, but also relatively poor resolution, groove, which normally leads from the labial segment to
withFastRedstainingandconfocalmicroscopy,whichpro- the tip of the collophore, ended in between the A1 legs
vides higher resolution, but also, in our hands, high back- (Figure1E,Figure4B;comparewithFigure4A).Intheless
ground (Figure 2). The specific shape of the A1 appendage stronglytransformedindividuals,thelegsonA1werefused
budsservedasamarkerfordistinguishingbetweenabdom- in the midline while still retaining their leg-like identity
inalsegmentsinyoungembryos. (Figure5);thissuggeststhatlowlevelsofOc-Ubxaresuffi-
InearlystagesofexpressionbothOc-UbxandOc-abd-A cient to promote appendage fusion, but higher levels are
were detected as two pairs of spots, which we identified required to transform the legs into the vesicular organ of
as buds on A1 and A3 for Oc-Ubx and A3 and A4 for thecollophore.
Oc-abd-A (Figure 2A,E). Expression of both genes was The ectopic furca on A3 resembled the normal furca
also visible in A2, but was less intense (Figure 2A,C). In on A4, except that even in the most strongly trans-
slightlyolderembryos,theexpressionspreadtootherparts formed individuals it was smaller (Figure 1E, Figure 3H;
ofthesegmentsandbecamemoreintense(Figure2B,F).In compare with Figure 3G). In summary, Oc-Ubx in
embryos with well-defined abdominal appendages, Oc-Ubx Orchesella is required for giving the appendage buds on
was evident in A1 to A3 and Oc-abd-A in A2 to A4 seg- A1thecollophoreidentityandthoseonA3theretinacu-
ments(Figure2D,G).Wecouldnotseewhethertheexpres- lum identity; a general function of Oc-Ubx is to promote
sion domains extended also to the posterior part of the thefusion ofpairedappendageprimordia.
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Figure2ExpressionofOc-UbxandOc-abd-AinOrchesellaembryos.BrightfieldimagesofNBT/BCIPstained(A,B,E,F)andconfocalimages
ofFastRedstained(C,D,G)Orchesellaembryos.(A-D)Oc-Ubxexpressionand(E-G)Oc-abd-Aexpression.(AandE)areventralviews,other
picturesarelateralviews,headatthetop.Orchesellaembryogenesislasts4.75days;theembryosin(A,C,E)are30h,in(B,F)32h,andin(D,G)
48hold.(A)TheearlysignsofOc-Ubxexpressionareseenastwopairsofdots(A1,A3),withaweaksmudgeinthemiddle(asterisk).(B)Inan
olderembryo,thestainingismoreintense,extendedlaterallyandslightlyanteriorly.ThestainingdoesnotspreadbehindA3.(C)Confocalimage
ofanembryoagedastheembryoin(A).Theintensityofstainingobtainedusingthistechniqueisstronger.Inall(A-C)theexpressionishighestinA1.
(D)Inanolderembryowithwell-developedabdominalappendages,Oc-UbxisdetectedinA1-A3.(E)TheexpressionofOc-abd-Aappearsastwopairs
ofdots(A3,A4).(F)Inaslightlyolderembryothestainingismoreintenseandspreadsonesegment(blackarrowhead)anteriorly.Staininginthedorsal
organ(do)isnotspecific.(G)Inanembryowithwell-developedabdominalappendages,Oc-abd-AisdetectedinA2toA4.A1toA4,thefirsttothe
fourthabdominalsegment;do,dorsalorgan;h,head;T1toT3,thefirsttothethirdthoracicsegment.Theinterpretationofappendageidentityisbased
ontheobservationofdozensofembryos;abdominalappendagescanbedistinguishedbytheirspecificmorphology.Scalebars:all100μm.
KnockdownofOc-abd-Aleadstohomeotic transformed into a collophore and the furca on A4 was
transformationoftheA3andA4appendages transformed into walking legs (Table 2). Strong trans-
We next repeated the injections with Oc-abd-A formations are illustrated in (Figure 1C and F). These
dsRNA. As with Oc-Ubx(RNAi), injection of Oc-abd-A larvae fed and walked until the end of the first instar,
(RNAi) had no effect on hatching. In over 75 per cent when most of them died. Only a few of the more
of the resulting larvae, the retinaculum on A3 was weakly affected individuals started ecdysis to the
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Table2EfficiencyofOc-UbxandOc-abd-AsilencingbyparentalRNAiinOrchesella
dsRNA Females Larvae Homeoticphenotype
collected
Injected Survived None Weak Strong
egfp 96 80(83%) 415 415(100%) − −
MalE 27 20(74%) 195 195(100%) − −
Oc-Ubx 77 52(67.5%) 249 58(23.5%) 44(17.5%) 147(59%)
Oc-abd-A 99 67(68%) 622 140(22.5%) 205(33%) 277(44.5%)
Oc-Ubx+MalE 27 12(44%) 98 41(42%) 15(15%) 42(43%)
Oc-abd-A+MalE 42 26(62%) 173 75(43.5%) 32(18.5%) 66(38%)
Oc-Ubx+Oc-abd-A(injectedonce) 49 37(76%) 141 7(5%) 107(76%) 27(19%)
Oc-Ubx+Oc-abd-A(injectedtwice) 102 47(46%) 43 4(9.5%) 6(14%) 33(76.5%)
Eggswerecollectedfor10daysafterfemaleinjections.Alllarvaehatched;wedidnotcounttheunfertilizedeggs,whichareoccasionallylaidbyRNAi,controland
non-injectedfemalesandalwaysrepresentlessthan1%.dsRNAsforegfpandMalEgenesservedascontrols.Thetableshowsonlyapartofourexperiments
wherewescoredandcountedallthelarvae.Intotal,weobservedoffspringfrom239,99,316,120femalesinjectedwithegfp,MaleE,Oc-UbxandOc-abd-AdsRNA,
respectively;wealwayssawonlythephenotypesspecificforeachtreatment.Homeoticphenotypes:strong:transformedappendagesmorphologicallysimilarto
theirnormalcounterparts;weak:appendageswithsignsoftransformation,butmalformed,forexample,A1andA4partiallyfusedproximallyinOc-Ubx(RNAi)or
Oc-abd-A(RNAi),A3blister-likeinOc-abd-A(RNAi),elongated,non-segmented,partiallyfusedproximallyinOc-Ubx+Oc-abd-A(RNAi).
second instar; these died during shedding the cuticle in A1 throughout A2 to the tip of the ectopic collo-
or soon after. phore on A3 and even behind it (Figure 4C; compare
The collophore on A3 of the most strongly trans- with Figure 4A,B); his suggests that in normal animals
formed individuals was similar in morphology and size one role of Oc-abd-A is to inhibit development of the
to the endogenous collophore on A1 (Figure 1C,F; com- ventral groove in segments posterior to A1.
pare with Figure 1D; Figure 3F; compare with Figure 3E). In summary, Oc-abd-A is required for specification of
The ectopic legs on A4 were segmented like legs; their the retinaculum on A3 and the furca on A4. It induces
proximalpartswereseparate(Figure1C,F)andtheyhada fusion of the appendages (similarly to Oc-Ubx) and in
claw at their tips (Figure 3D; compare with Figure 3A,C). the cuticle it represses the ventral groove. In the absence
Theywerenotmovedduringwalking.The ventral groove of Oc-abd-A, both the A3 and the A4 appendages trans-
was extended from its normal ending at the collophore form toamoreanteriorfate.
Figure3Detailsofthehomeoticallytransformedappendages.A-D.ThehomeoticlegsonA1(B)andA4(D)haveaterminalclawlikethe
controlwalkinglegsonT3(A);theclawsaremarkedwithwhitearrows.Notethedifferencebetween(B)andthecontrolcollophoreonA1in(E),and
between(D)andthetipofthecontrolfurcaonA4in(C).Thetwoappendagesformingthefurcaterminatewithspecializedstructures(mucrones),
markedherewithblackarrowheads;bothappendagesareshown.(E,F)ThecollophoreonA3(F)resemblesthecontrolcollophoreonA1byitsgross
morphologyandthepresenceofcharacteristicsmoothcuticleatitstip(whitearrowheads).(G,H)ThehomeoticfurcaonA3has,likethecontrolfurca
onA4,theproximalcommonmanubrium(m)bifurcatedintotwodistaldentes(d),althoughitissmaller(comparethescalebars).Mostofthesetae
onthehomeoticfurcain(H)werelostduringsamplepreparation.NotehowtheA3homeoticappendagesin(F)and(H)differfromthecontrol
retinaculumonA3in(Figure1D).Scalebars:inA,20μmfor(A-D);inE,20μmfor(E,F);(G)100μm;(F)50μm.
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Figure5WeakOc-Ubx(RNAi)phenotype.LowlevelsofOc-Ubx
aresufficientforappendagefusion,butdonotprevent
developmentofwalkinglegmorphology.TheappendagesonA1of
thislarvaresemblethelegsandhaveclawsattheirtips(yellow
arrowsandinset;comparewiththeclawatthetipofthethoracic
legmarkedwithablackarrowhead),buttheirproximalpartsare
fused(asterisk).Thewhitearrowmarkstheventralgroove.A1,T2,T3
markthefirstabdominal,thesecondandthethirdthoraciclegs,
respectively.Scalebar:50μm.
appendages in Oc-abd-A(RNAi). We next wanted to test
whether simultaneous silencing of Oc-Ubx and Oc-abd-A
bydouble RNAi wouldtransform all Orchesella abdominal
appendagesintolegs,andwhetheritwouldallowthedevel-
opment of legs on A2, which normally makes no
appendage.
Effectiveknockdownoftwogenesisdifficult,becausethe
maximum amount of each type of dsRNA that can be
injected is reduced to one half compared with the single-
gene RNAi. To probe the strength of double RNAi in
Figure4Oc-abd-Ainhibitstheventralgroove.(A)Inwildtype Orchesella,wefirstco-injectedmotherswithdsRNAagainst
(andcontrol)embryos,theventralgroove(whitearrows)isachannel oneoftheHoxgenesmixedwithcontroldsRNA.Thepene-
inthecuticlethatrunsontheventralsideofthethoraxfromthehead
trance of the phenotype was slightly reduced for both Oc-
tothetipofthecollophore(c)onA1andneverextendsbehind.
Ubx and Oc-abd-A, but the strong transformation was still
(B)IntheOc-Ubx(RNAi)larvae,whichhaveapairofwalkinglegsonA1
insteadofthecollophore,theventralgroove(whitearrows)terminates observedinmorethan38percentoflarvae(Table2).
betweenthehomeoticwalkinglegs(asterisk).(C)IntheOc-abd-A When we injected the females with both Oc-Ubx and
(RNAi)larvae,theventralgroove(whitearrows)leadstothehomeotic Oc-abd-AdsRNAstogether,someoftheirprogenyhadall
collophoreonA3andevenextendsbehind.A1andA3markthefirst
of their abdominal appendages transformed towards the
andthethirdabdominalsegments,respectively.c,collophore;f,furca;
legidentity(Figure6).Thefrequencyofthestrongestphe-
r,retinaculum;wl,walkingleg.Thehomeoticappendagesarelabelled
inyellow.Scalebars:(A)100μm;(B)50μm;(C)20μm. notypeswashigherwhenthematernalinjectionswerere-
peated on two successive days (19 per cent after one
versus 76.5 per cent after two injections) but the number
SimultaneousknockdownofOc-UbxandOc-abd-A ofeggslaidbythetwice-injectedfemaleswaslower.More
transformsallOrchesellaabdominalappendagesintoleg-like than two consecutive injections led to lethality in the fe-
structuresbutdoesnotinduceappendagesonA2 males without production of eggs. In the most strongly
TheexperimentsaboveshowthatwhenOrchesellaabdom- transformedindividuals,thelegsonA1andA4wereiden-
inal appendages are missing the function of both Oc-Ubx ticalwiththosethatweobservedinthesingle-geneRNAi.
and Oc-abd-A they have a walking leg identity. We ob- The appendages on A3 were segmented like legs and had
servedthisintheA1appendagesinOc-Ubx(RNAi)andA4 their proximal parts separated, but remained short and
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Figure6DoubleOc-Ubx+Oc-abd-A(RNAi)phenotype.Larvathat
hadbothOc-UbxandOc-abd-AknockeddownbyparentalRNAihas
legsonA1(whitearrowheads)andA4(blackarrowheads)asinthe
single-geneRNAi.TheA3appendages(yellowarrows)alsoresemble
legs,butaremissingterminalparts.ThisA3‘leg’isenlargedinthe
inset;theasteriskmarksthedistaltiplackingtheclaw.A1,A3andA4
markthefirst,thethirdandthefourthabdominalsegments,respectively.
Scalebar:200μm.
Figure7SchematicsummaryofOc-UbxandOc-abd-A
knockdownexperimentsinOrchesella.Theboxesrepresentthe
thoracic(T1toT3)andabdominal(A1toA6)segments.Blueandgrey
lacked claws (Figure 6, inset). No appendages developed shadingrepresentOc-UbxexpressionandOc-abd-Aexpression,
onA2. respectively(examinedbyin-situhybridizationonwildtype,controland
knockdownembryos).TheappendagebudsinwhichonlyOc-Ubxis
In summary, these experiments show that double
expresseddevelopintothecollophore,thebudsinwhichonlyOc-abd-A
RNAi is possible in Orchesella, because we achieved
isexpresseddevelopintothefurca,andthebudsinwhichbothOc-Ubx
a novel transformation compared with single-gene andOc-abd-Aareexpresseddevelopintotheretinaculum.Whenneither
RNAi. Oc-UbxnorOc-abd-Aisexpressed,theappendagehasalegidentity.The
appendagesonA3aresmaller,suchasthefurcainOc-abd-A(RNAi),and
maybemissingdistalparts,suchasthe‘legs’inthedoubleOc-Ubx+
Discussion
Oc-abd-A(RNAi).TheA2segmentneverdevelopsappendagebuds.
In this study we have investigated the role of the Hox
wt,wildtype.
genes Ubx and abd-A in the specification of abdominal
appendages in the springtail Orchesella. The results of
ourRNAiexperimentsaresummarizeddiagrammatically Oc-UbxspecifiesauniqueidentityforA1inhexapods
inFigure7.We conclude that: In collembolans, as in insects, A1 is the only abdominal
segment thatisspecified by Ubx inthe absence ofabd-A.
(cid:1) Theactivity ofOc-Ubxalone causesappendage SilencingofOc-UbxinOrchesella replaces the collophore
budstodevelop asacollophore,because(i) with a pair of walking legs. Similarly in insects, depletion
when Oc-Ubx isknockeddown,thebudson ofUbxaloneresultsintheappearanceoflegstructureson
A1,whichexpressOc-Ubx butnotOc-abd-A, A1 [12,26,38,53,54]. In insects, in the absence of Ubx, ex-
developinto legs,notacollophore;(ii) when pression of the more anterior Hox gene Antennapedia
Oc-abd-Ais knocked down, thebuds onA3,which persistsinA1,whereitisnormallyrepressedbyUbx[55],
normallyexpressboth Oc-Ubx and Oc-abd-A, resulting in the development of a leg-like appendage. It is
developinto acollophore,presumablybecause likely that the same happens in collembolans, although
depletion ofOc-abd-AleavesOc-Ubx alone thishasnotbeentested.
expressed inthesebuds. In dipterans and some other endopterygote insects, for
(cid:1) TheactivityofOc-abd-Aalonecausesappendage example, the lepidopteran Manduca [56], Ubx functions
buds to develop as a furca, because (i) when asalimbrepressor,andA1bearsnoappendages.Inmany
Oc-abd-A is knocked down, the buds on A4, other species though, including the beetle Tribolium, the
which express only Oc-abd-A, develop into legs, milkweed bug Oncopeltus and the cricket Acheta, Ubx
not the furca; (ii) when Oc-Ubx is knocked down, specifies the embryonic glandular appendages on A1
so that presumably Oc-abd-A alone is expressed called pleuropods [26,37,38,53]. Like the collophore of
in A3, the A3 buds develop into a furca. springtails, pleuropods are vesicular structures that are
(cid:1) IfbothOc-UbxandOc-abd-Aareexpressedinapair likely involved in secretion or excretion, but unlike the
oflimbbuds,itdevelopsasaretinaculum. collophore,theyremainpairedratherthanformingasingle