The Functions of Grainy Head-Like Proteins in Animals and Fungi and the Evolution of Apical ...

26 Pages·2012·3.22 MB·English

The Functions of Grainy Head-Like Proteins in Animals
and Fungi and the Evolution of Apical Extracellular
Barriers
Adam Pare´1*, Myungjin Kim1, Michelle T. Juarez1, Stuart Brody2, William McGinnis1*
1SectionofCell&DevelopmentalBiology,UniversityofCaliforniaSanDiego,LaJolla,California,UnitedStatesofAmerica,2SectionofMolecularBiology,Universityof
CaliforniaSanDiego,LaJolla,California,UnitedStatesofAmerica
Abstract
TheGrainyhead(GRH)familyoftranscriptionfactorsarecrucialforthedevelopmentandrepairofepidermalbarriersinall
animalsinwhichtheyhavebeenstudied.Thisisahigh-levelfunctionalconservation,astheknownstructuralandenzymatic
genes regulated by GRH proteins differ between species depending on the type of epidermal barrier being formed.
Interestingly, members of the CP2 superfamily of transcription factors, which encompasses the GRH and LSF families in
animals,arealsofoundinfungi–organismsthatlackepidermaltissues.ToshedlightonCP2proteinfunctioninfungi,we
characterized a Neurospora crassa mutant lacking the CP2 member we refer to as grainy head-like (grhl). We show that
NeurosporaGRHLhasaDNA-bindingspecificitysimilartothatofanimalGRHproteinsanddissimilartothatofanimalLSF
proteins.Neurosporagrhlmutantsaredefectiveinconidial-sporedispersalduetoaninabilitytoremodelthecellwall,and
weshowthatgrhlmutantsandthelong-knownconidialseparation-2(csp-2)mutantsareallelic.Wethencharacterizedthe
transcriptomesofbothNeurosporagrhlmutantsandDrosophilagrhmutantembryostolookforsimilaritiesintheaffected
genes.Neurosporagrhlappearstoplayaroleinthedevelopmentandremodelingofthecellwall,aswellasintheactivation
ofgenesinvolvedindefenseandvirulence.DrosophilaGRHisrequiredtoactivatetheexpressionofmanygenesinvolvedin
cuticular/epidermal-barrierformation.WealsopresentevidencethatGRHplaysaroleinadultantimicrobialdefense.These
results,alongwithpreviousstudiesofanimalGRHproteins,suggestthefascinatingpossibilitythattheapicalextracellular
barriersofsomeanimalsandfungimightshareanevolutionaryconnection,andthattheformationofphysicalbarriersin
the lastcommonancestorwasunderthe control ofatranscriptional code thatincludedGRH-like proteins.
Citation:Pare´A,KimM,JuarezMT,BrodyS,McGinnisW(2012)TheFunctionsofGrainyHead-LikeProteinsinAnimalsandFungiandtheEvolutionofApical
ExtracellularBarriers.PLoSONE7(5):e36254.doi:10.1371/journal.pone.0036254
Editor:JasonE.Stajich,UniversityofCaliforniaRiverside,UnitedStatesofAmerica
ReceivedDecember15,2011;AcceptedApril3,2012;PublishedMay9,2012
Copyright:(cid:2)2012Pare´etal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted
use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisresearchwassupportedbyNationalInstitutesofHealthGrants(http://grants.nih.gov/)GM077197andHD28315(toWM),NIHTrainingGrant
T32GM007240(toAP)andalsoreceivedsupportfromthefamilyofHerbertStern.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected](WM);[email protected](AP)
Introduction proteins in the Drosophila cuticle [1,3]. Furthermore, grh embryos
arepermeabletoexogenouslyapplieddyes[6],andtheremovalof
Grainy head (GRH) transcription factors are crucial for many GRH from imaginal disc cells results in reduced expression of at
aspectsofdevelopment.Forinstance,DrosophilaGRH(alsocalled leasttwocell-adhesiongenes[13].Thesefindingssuggestthatthe
Elf-1orNTF-1)regulatesdevelopmentoftheepidermisandhead paracellularintegrityoftheepithelialbarrierunderlyingthecuticle
skeleton[1,2],woundhealing[3–6],neuroblastproliferation[7,8], becomescompromisedinDrosophilagrhmutants.Inadditiontothe
earlyembryonicpatterning[9,10],andtracheal-tubemorphology developmentalfunctionsofGRHinDrosophila,itisalsonecessary
[11].However,thefunctionsofGRHfamilyproteinswithrespect fortheproperexpressionofseveralcuticular-barriergenesthatare
to epidermal-barrier formation andwound healing have received activated during the regenerative process following epidermal
the most attention, as these functions appear to be widely wounding[3–5].
conserved inanimals. GRH family proteins are also important for epidermal-barrier
Drosophilagrhmutantembryoshaveslackandfragilecuticles,as formation in the distantly related invertebrate C. elegans. RNAi
well as ‘‘grainy’’ and discontinuous head skeletons [1,2,12]. Null targeted against Ce-Grh-1 results in embryos with a fragile and
mutations are lethal, as the embryos fail to develop past the puckered hypodermis – a similar phenotype to that seen in
embryonic/larval transition point due to their extremely fragile Drosophila [14]. Ce-Grh-1 binds the same palindromic consensus
epidermal barriers. These phenotypes clearly point to defects in DNAsequencesasDrosophilaGRH,andtheDdcgeneinC.elegans
the formation of chitin-based cuticular structures in grh mutant has GRH binding sites upstream of its promoter [14]. Strikingly,
embryos. These defects are likely due to lowered epidermal conservationofGRHfamilytranscriptionfactorfunctionextends
expression of a wide variety of genes, among them Ddc, which to vertebrates as well, despite vast differences in the structural
encodesdopadecarboxylase,anenzymerequiredtogeneratethe componentsofepidermalbarriersbetweenandwithinprotostome
reactive quinone molecules used to cross-link chitin fibers and and deuterostome animals. In Xenopus laevis, expression of a
PLoSONE | www.plosone.org 1 May2012 | Volume 7 | Issue 5 | e36254
GrainyHead-LikeFunctioninAnimalsandFungi
dominant negative form of XGRHL1 leads to a malformed outmicroarrayandphenotypicanalysesofDrosophilagrhmutants,
epidermis, partly due to lowered expression of keratin [15]. The and we present evidence that, in addition to its crucial role in
mouse genome contains three paralogs of GRH, encoded by the cuticular- and epidermal-barrier formation, GRH may also be
genesGrainyhead-like1,22,and23(Grhl1,22,and23),whichare involvedinmicrobialdefenseduringadulthoodinDrosophila.Our
allexpressedinthesurfaceectodermduringdevelopment[16–18]. results suggest an ancestral role for CP2 superfamily proteins as
Mutations of both mouse Grhl1 and Grhl3 genes result in a regulatorsofextracellular-barrierformationinopisthokontances-
malformedepidermis.Grhl3(alsoknownasGet1)knockoutmutants tors.
displaythemostseverephenotypes,includingabnormalepithelial
morphology in both the epidermis and bladder, impaired wound Results
healing, defective extracellular lipid processing, increased perme-
abilitytoexogenousdyes,andseverepostnatalwaterloss,aswell Sequence Analyses Suggest Fungal CP2 Proteins are
as defects in neural-tube and eyelid closure [16,19–24]. Grhl1- MoreFunctionallySimilartoAnimalGRHProteinsthanto
deficient mice display delayed coat growth, thickened paw skin, Animal LSF Proteins
and hair loss due to poor anchoring of hair shafts within follicles The CP2 superfamily is composed of the GRH and LSF
[25]. Grhl2 appears to regulate neural-tube closure as well as E- families of transcription factors. A comprehensive review of the
cadherin expression [26]. Furthermore, all three mouse Grhl functions of LSF-like proteins is beyond the scope of this paper,
transcriptionfactorshavebeenshowntobindpreferentiallytothe butthereappearstobelittleoverlapbetweenthebiologicalrolesof
sameconsensusDNAsequencesasDrosophilaandC.elegansGRH the GRH and LSF families in animals [29],and the two families
proteins [27]. havedivergedgreatlyintheirmodesofDNAbinding[30,31].Itis
WhiletheDNA-bindingspecificityofGRHfamilyproteinshas clear that the last common ancestor of Metazoa and Fungi
been conserved between protostome and deuterostome animals, possessed at least one CP2 superfamily protein, although
thedownstreameffectorsofGRH-likeproteinsindistantlyrelated phylogenetic analysis indicates that fungal CP2 superfamily
species do not appear to be homologous, but instead carry out proteins form a separate outgroup with respect to metazoan
analogous functions suited to the specific barrier being generated GRH and LSF family proteins [28]. With few exceptions, all
orregeneratedafterwounding.Forinstance,theepidermaldefects sequencedmetazoangenomespossessoneormorecopiesofboth
in Grhl3-deficient mice correlate with reduced levels of transgluta- GRH and LSF family proteins. Among the Fungi, only
minase1transcription(whichhasupstreamGRHbindingsites),as ascomycete and zygomycete genomes encode a CP2 superfamily
well as reduced transcription for many genes that are structural protein (or multiple paralogs), while known basidiomycete
barrier components of differentiated corneocytes [19,21]. Trans- genomes do not. Some ascomycetes (e.g., Saccharomyces cerevisiae)
glutaminase 1 is an enzyme necessary for the cross-linking of appear to have lost the CP2 superfamily. The unicellular sister-
keratin and other proteins in the mammalian epidermis, and it grouporganismsM.brevicollis(achoanoflagellate)andC.owczarzaki
plays an analogous role to that of dopa decarboxylase in the (a filasterean) both contain single CP2 superfamily proteins
Drosophila cuticle. In sum, there exists a high-level functional [32,33].
conservationofGRHproteinsasregulatorsofepidermalintegrity Althougha recently published phylogenetic analysisusing gap-
andwoundhealinginbothprotostomeanddeuterostomeanimals free alignments of near full-length protein sequences showedthat
(whichdivergedapproximately700millionyearsago),despitethe fungal CP2 superfamily proteins are roughly equally related to
significant structural differences in barrier composition across the boththeGRHandLSFproteinfamilies[28],wedecidedtolook
animal kingdom. This functional conservation is reminiscent of more closely at the DNA-binding domain sequences of extant
other cases in which high-level transcription factor function has opisthokont CP2 superfamily proteins to identify specific residues
been conserved over great evolutionary time (e.g., Hox genes, that might be characteristic of GRH or LSF proteins. An
Pax6/eyeless, and Nkx2.5/tinman in body-axis, eye, and heart alignmentbetweentheDNA-bindingdomainsoftwoGRHfamily
specification,respectively)despitethedriftofspecificdownstream proteins (D. melanogaster GRH and H. sapiens Grhl1), two LSF
effectors. family proteins (D. melanogaster GEM and H. sapiens LSF), and a
Since the function of GRH-like proteins in epidermal-barrier representative fungal CP2 superfamily protein (referred to as
formation and wound healing appears well conserved in triplo- Neurospora Grainy head-like, or GRHL, for reasons described
blastic animals, we were interested in determining what role below)highlightstheextensivesequence conservation throughout
GRH-like proteins might be playing in more distantly related thisdomain(Figures1AandB).Ithasbeenpredictedthatpartof
organisms. GRH family proteins (along with the related LSF the region containing the DNA-binding domain of CP2 super-
family proteins) belong to the CP2 superfamily of transcription family proteins adopts a similar tertiary structure to the DNA-
factors, members of which are only found in the opisthokont bindingdomainofp53[34],whichhasawell-characterizedthree-
lineage, which includes Metazoa (Animals), Fungi, and several dimensional structure.Strikingly,theidentityofeightaminoacid
closely related sister-species [28]. Considering the fact that Fungi residuesatandaroundpositionspredictedtobecrucialforDNA
utilizeaverydifferenttypeofextracellularphysicalbarrier(thecell binding, based on mapping to the p53 structure (i.e., major- and
wall)comparedwithanimals,wethoughtthatbystudyingtherole minor-groove contacts, zinc-binding residues, and residues
of CP2 superfamily transcription factors in Fungi we might shed involvedindimerization)suggestthattheDNA-bindingproperties
some light on the origins of transcriptional control of physical- offungalCP2proteinsmightbemoresimilartoGRHthantoLSF
barrier formation in the opisthokont ancestor. Towards this end, familyproteins.Forexample,relativetopositions194–198ofthe
wehavecharacterizedthefunctionoftheCP2superfamilygenein NeurosporaGRHLDNA-bindingdomain(aregionpredictedtobe
the ascomycete fungus Neurospora crassa using microarray and involved in major-groove interaction) the same amino acid
phenotypicanalyses.WeshowthatthelossofthisNeurosporagene, sequence GAERK is found in nearly all available metazoan
whichwecallgrainyhead-like(grhl),leadstoadevelopmentaldefect GRH and fungal GRHL ortholog sequences, while the sequence
in cell wall remodeling during conidial development, which is GADRK is found in all available metazoan LSF sequences
associatedwiththedown-regulationofnumerousgenespredicted (Figure 1B). Similarly, in three other regions predicted to be
toencode abundantcomponentsofthecellwall.Wealsocarried important for DNA binding (positions 81–84, 142–147 and 150–
PLoSONE | www.plosone.org 2 May2012 | Volume 7 | Issue 5 | e36254
GrainyHead-LikeFunctioninAnimalsandFungi
153),wefindthatsomeNeurosporaGRHLaminoacidresiduesare domains–animalGRHproteinsalsolackSAMdomains,butall
identical to those found in nearly all fungal CP2 proteins and knownanimal LSFfamilyproteins possessSAMdomains[28].
metazoanGRHproteins,buttheydifferfromthosefoundinLSF
proteins (indicated with asterisks in Figure 1B). Based on these The Neurospora CP2 Protein GRHL and Drosophila GRH
observations, we hypothesized that the DNA-binding character- have Similar DNA-binding Specificities
istics of the Neurospora GRHL protein might be more similar to
Totest whether a fungal CP2proteinsbinds DNAsimilarly to
those of animal GRH family proteins than animal LSF family
animal GRH family proteins, we decided to study the Neurospora
proteins.AnotherindicationthatfungalCP2superfamilyproteins
crassaCP2superfamilyprotein.WechoseNeurosporaasourmodel
mightbemorefunctionallysimilartoanimalGRHproteinsisthat
organism because it is a fairly typical representative of a
fungal CP2 superfamily proteins all lack SAM oligomerization
filamentousascomycetefungus,thereexistanumberofmolecular
tools to work with (including gene-knockout technologies), and it
Figure1.NeurosporaGRHLhasasimilarDNAbindingspecificityasDrosophilaGRH.(A)TheNeurosporaGRHLproteinsharessequence
similaritywithbothDrosophilaGRHandmammalianGrhlproteins,aswellaswithLSFfamilyproteins[28].Theareasofhighestsimilarityincludethe
regioncontainingtheGRHDNA-bindingdomain,andaregionneartheC-terminuscontainingtheGRHdimerizationdomain[30].(B)Acomparison
oftheDNA-bindingdomainofarepresentativefungalCP2superfamilyprotein(N.craGRHL)withthoseoftheDrosophilaandhumanGRHfamily
proteins(D.melGRHandH.sapGrhl1)andLSFfamilyproteins(D.melGEMandH.sapLSF).AminoacidresiduespredictedtobeimportantforDNA
interactionsbasedoncomparisonswithp53transcriptionfactors[34]aremarkedabovethealignmentasfollows:‘‘D’’–dimerization;‘‘Z’’–zinc-
binding;‘‘m’’–minor-grooveinteraction;and‘‘M’’–major-grooveinteraction.ResiduesthatdistinguishGRHfamilyfromLSFfamilyproteinsare
indicatedbelowthealignmentwithasterisks.TheindicatedaminoacidsinnearlyallknownfungalCP2superfamilyproteinsareidenticaltothe
Neurosporaresidues.(C)Oligonucleotidesusedinthegel-shifts.BasesthatincludetheLSForGRHoptimalconsensusbindingsitesareindicatedwith
asterisks.(D)Gel-shiftassaystestingDrosophilaGRHandNeurosporaGRHLbindingtotheoligonucleotidesin(C).Thebottompanelswereexposed
for 16.5h, and the top panels were exposed for 75h. Specific bands are indicated with black arrowheads, and weak specific bands are also
highlighted with asterisks in the top panels. Nonspecific (NS) bands were also detected in the no-protein-template negative controls (data not
shown),andtheyareindicatedwithwhitearrowheadsinthetoppanelsandwithabarinthebottompanels.
doi:10.1371/journal.pone.0036254.g001
PLoSONE | www.plosone.org 3 May2012 | Volume 7 | Issue 5 | e36254
GrainyHead-LikeFunctioninAnimalsandFungi
has a fully sequenced genome. The Neurospora crassa genome rhythm) aerial hyphae grow up and away from the food source
possesses a single CP2 superfamily gene (designated as andbudoffchainsofnewconidia.Theseconidialchainsbecome
NCU06095), which has been called grainy-head homolog (ghh) [35], quite delicate as they mature, as the thick cross-walls between
butwhichwewillhereafterrefertoasgrainyhead-like(grhl)orcsp-2, individual conidia are remodeled into thin, easily broken
forthereasonsdescribedintheprecedingandfollowingsections. connectives – this allows mature conidia to readily detach and
Using RT-PCR and primers specific to the predicted start and disperse tofound newcolonies.
stop sites, we cloned and sequenced the full-length grhl coding Neurospora strains containing precise deletions of the entire grhl
regionandfoundthesequenceandexonstructuretobeidentical coding region were obtained from the Fungal Genetics Stock
to that in the Broad Institute Neurospora database. No splice Center (FGSC) for both mating type (mat) backgrounds:
variantsweredetected,althoughwecannotruleoutthepossibility FGSC13563 (mat A) and FGSC13564 (mat a). In addition, we
of grhl transcripts that include additional upstream exons or createdmultipleindependentlyderivedgrhlknockoutstrainsusing
alternate 3’UTRs. targetedhomologousrecombinationtoreplacethegrhllocuswitha
Wesynthesizedfull-lengthNeurosporaGRHLproteininorderto hygromycincassette.Thephenotypesofthesemutantstrainswere
characterizeitsDNA-bindingpropertiesusinggel-shiftanalyses.It indistinguishablefromthoseofthedeletionmutantstocksobtained
hasbeenshownthatDrosophilaandC.elegansGRHfamilyproteins from the FGSC, indicating that the phenotypes described below
canbothbindwithhighaffinityashomodimerstothepalindromic areindeedduetothelossofgrhlfunction.PCRamplificationofa
DNA sequence ACCGGTT from the Ddc promoter [14,30] and region within the grhl locus verified that all strains were indeed
that the optimal consensus binding site for murine GRH family lacking the grhl gene (Figure 2A). Furthermore, RT-PCR
proteins contains the palindromic DNA sequence AACCGGTT amplification of a region of the grhl mRNA yielded no product
[19,27].MammalianLSFhasbeenshowntobindasatetramerto when RNA from grhl mutants was used as template, compared
DNAfragmentscontainingthesequenceCTGG-N -CTGG;LSF with robust detection of grhl transcripts using wild-type RNA as
6
doesnotbindtoDNAfragmentscontainingaGRHsitefromthe template (Figure 2B). Transcripts from the grhl gene were readily
Ubx promoter [31]. On the other hand, Drosophila GRH can detectable by RT-PCR using wild-type RNA templates from
weakly bind to both full-length LSF sites and CTGG half-sites either pure mycelial samples or samples of aerial hyphae and
[31]. Therefore, we tested the ability of the Neurospora GRHL conidia(thelatteryieldingslightlystrongeramplifications;datanot
protein to bind DNA oligonucleotides containing one of the shown),whichsuggeststheGRHLtranscriptionfactorisexpressed
following sites: the endogenous GRH binding site (GRH-Ddc), a inmost Neurospora celltypesduring asexual proliferation.
mutated GRH binding site (GRH-mut), or the consensus GRH Mutant grhl strains areviable andcan bepropagated asexually
binding site (GRH-con) (Figure 1C). We also tested the ability of ashomokaryoniccolonies(i.e.,allnucleiinthecolonyareclonal)
theGRHLproteintobindDNA oligonucleotidescontaining one onminimalmedia.Bothgrhlmating-typestrainscanserveasmales
of the following sites: the endogenous LSF consensus site (LSF- or females in sexual crosses to wild-type or grhl strains of the
con),anLSFhalf-site(LSF-1/2),oramutatedLSFsite(LSF-mut) opposite mating type, indicating that grhl function is dispensable
(Figure1C).Thebindingoffull-lengthDrosophilaGRHproteinto for sexual reproduction (data not shown). The grhl strains appear
these oligonucleotides wastested asa comparison. quite healthy and in many ways are indistinguishable from wild
Drosophila GRH bound DNA sequences as previously reported type, atleast underlaboratory conditions(Figures 2D–F).
[30,31], interacting strongly with the GRH-Ddc and GRH-con The grhl mutant strains display a slightly altered circadian
oligonucleotides, but not with the GRH-mut oligonucleotide rhythm [35], develop orange pigmentation slightly more quickly
(Figure1D,rightpanels).DrosophilaGRHalsoboundveryweakly thanwildtype(Figure2C),andsometimeshavepalermyceliathan
toboththeLSF-conandLSF-1/2oligonucleotides,butnottothe wildtype(datanotshown).However,themoststrikingphenotype
mutated LSF-mut oligonucleotide (Figure 1D, top right panel). ofgrhlmutantsisapronounced conidial-separationdefect.Ingrhl
Neurospora GRHL bound with a similar specificity as Drosophila strains, conidial chains fail to completely separate, even upon
GRH,albeitwithapparentloweraffinity.GRHLboundstrongly physical stress or immersion in liquid (Figures 2G–I). This
to the GRH-con oligonucleotide, weakly to the GRH-Ddc phenotype is identical to that observed in the conidial separation
oligonucleotide, and very weakly to the LSF-con oligonucleotide mutantscsp-1andcsp-2,whosephenotypeshavebeeninvestigated
(Figure 1D, left panels). Considering these results, along with the in some detail. It was shown that the csp mutant strains begin
similaritiesintheirDNA-bindingdomainsequences,weconclude conidialdevelopmentnormally;however,thechitinouscross-walls
that the last common ancestor of opisthokonts possessed a CP2 between adjacent conidia do not become remodeled into thin
superfamily protein with a similar DNA-binding specificity to connectives, precluding conidial separation [37]. This phenotype
existing metazoan GRHfamily proteins. was correlated with a decrease in the autocatalytic activity of the
Neurosporacellwall,whichwashypothesizedtobeduetothelossof
Phenotypes of the Neurospora grhl Knockout Mutants secreted enzymessuch aschitinase [38].
The fungus Neurospora crassa has a simple cellular organization While csp-1 and csp-2 have long been popular background
and life cycle compared with most animals and plants (for an in- strains for Neurospora researchers (they help prevent the cross-
depth treatment on the subject, see [36]). The most visually contamination of stocks), the nature of the mutant genes
obviousphaseoftheNeurosporalifecycleisasexualproliferation– responsible for these phenotypes remained unknown for many
single spores (conidia) germinate on a food source and form a years. Recently, it was shown that csp-1 (NCU02713) encodes a
densely interwoven mat of thread-like mycelia, which spreads zinc-finger transcription factor on chromosome 1 [39]. However,
quickly to form a colony. Neurospora colonies exist as syncytial nature of the gene underlying the csp-2 phenotype remained
collectionsof‘‘cells’’whichshareacommonextracellularbarrier– unclear, except that it mapped to chromosome 7 between the
the cell wall. While there are regularly spaced septa along the genes thi-3 and ace-8 [40] – precisely the region where grhl is
length of the mycelial and hyphal axes, these divisions are not located.Therefore,webelievedthatalesioninthegrhlgenemight
complete, and the ‘‘cells’’ use vigorous cytoplasmic streaming to beresponsible forthe csp-2 phenotype.
movenutrientsandothermoleculesthroughoutthecolony.After We first carried out genetic complementation tests with the
about a day (and every day after that, according to a circadian recessiveallelestotestwhethercsp-2andgrhlareallelic.Different
PLoSONE | www.plosone.org 4 May2012 | Volume 7 | Issue 5 | e36254
GrainyHead-LikeFunctioninAnimalsandFungi
Figure 2.Neurosporagrhlmutants display a conidial-separation phenotype. (A) PCR verification that the grhl locus is deleted in two
independentlygeneratedgrhlknockoutstrains(grhl-1andgrhl-2)andinaknockoutobtainedfromtheFGSC(grhl).Theactinlocuswasamplifiedasa
control.(B)RT-PCRdemonstratesthatgrhltranscriptsarenotbeingproducedintheFGSCgrhlknockoutstrains(matingtypesaorA).Similarresults
wereobtainedusingtotalRNAfromconidiaormycelia.Transcriptsfromtheactingenewereamplifiedasacontrol.(C)Mutantgrhlstrainsreachfull
pigmentation more quickly than wild type. Colonies were grown on Petri dishes for 48h at room temperature in a 12h light/dark cycle.
Approximately4cm2ofeachmatureconidiatingcolonyareshownhere.(D–F)Neurosporagrhlandwild-typestrainshavesimilargrowthratesand
eventuallyreachequalpigmentationlevels.ShownareVogel’sagarslantswithcolonygrowthafter5daysat30uCinconstantlight(D),a12hlight/
darkcycle(E),orconstantdark(F).(G–I)Neurosporagrhlstrainsdisplayapronouncedconidial-separationphenotype.(G)Wild-typeconidialchains
readilydisperseinglyceroltoyieldindividualspores.(H)Mutantgrhlconidialchainsremainintactinglycerol,evenaftervigorousagitation.(I)A
close-upviewofagrhlconidialchaindemonstratingthattheconidiaremainconnectedbythickconnectives(arrows).
doi:10.1371/journal.pone.0036254.g002
Neurospora strains can fuse to form syncytial heterokaryonic MicroarrayProfilingofNeurosporagrhlKnockoutMutants
colonies containing nuclei from both parental strains; these fused Todeterminethegenesdirectlyandindirectlyunderthecontrol
colonies are often able to grow in conditions that their parents of GRHL in Neurospora, we carried out microarray-based
cannot,aseachtypeofnucleuswillcomplementtherequirements transcriptome profiling of three different sample types: 1) MYC
oftheother.Forinstance,fusedcoloniesfromdifferentnutritional- –activelygrowingpuremycelialsamples;2)AHC–aerialhyphae
auxotroph parents can survive on minimal media, which can be and conidia from 48h old colonies; and 3) ALL – all cell types
takenadvantageoftotestforgeneticcomplementationatanother from 48h old colonies. We only describe the results from the
non-selectable locus. Using standard sexual-crossing procedures, AHC samples, as these are the cell types that displayed the
csp-2 and grhl mutations were placed into different auxotrophic conidial-separation phenotype (see the Materials and Methods
backgrounds (inos and his-3, respectively), and conidia from each section for the accession numbers of the MYC and ALL
strainwerecombinedonminimalmedia.Wefoundthatallviable microarraydatasets).
heterokaryonic fusings resulted in colonies that still displayed the Of the 10,526 genes that were probed on the microarray, 167
conidial-separation phenotype, demonstrating that csp-2 and grhl were seen to be misregulated in the grhl AHC samples at a False
mutantallelesfailtocomplement(seeMaterialsandMethodsfor DiscoveryRate(FDR)thresholdoflessthan0.01(meaning1%,or
details). To assay for the basis of the non-complementation, we about 2 of these genes, are expected to be false positives). This
sequencedthegrhlopenreadingframeofthecsp-2[FS590]allele,and thresholdroughlycorrespondstoagreaterthantwofoldchangein
found a one base pair deletion in codon S509. This mutation expression up or down relative to wild-type levels. Nearly equal
would be predicted to result in a premature stop codon after 14 numbersofgeneswereseentobeup-ordown-regulated(84and
out-of-framecodons,leadingtotheremovaloftheproper286C- 83genes,respectively),andverificationofmicroarrayfold-change
terminalaminoacidsoftheGRHLprotein(FigureS1).Therefore, directionalityfortengenesusingquantitativeRT-PCRisshownin
we conclude that grhl and csp-2 are allelic and that the conidial- Figure S2. Up-regulated genes on the grhl AHC microarrays are
separationphenotypeobservedingrhlstrainsisduetoareduction shown in Figure S3, the largest classes of which include genes
in the autocatalytic activity of the cell wall [38], which in turn involved in nitrogen, sulfur, and selenium metabolism, as well as
precludes remodeling of the cross-walls between adjacent conidia genesinvolvedinmembranetransportandcellularimport.Aswe
[37]. were interested in finding commonalities between the gene
PLoSONE | www.plosone.org 5 May2012 | Volume 7 | Issue 5 | e36254
GrainyHead-LikeFunctioninAnimalsandFungi
products activated by GRH-like transcription factors in animals Strikingly,themoststronglydown-regulatedgeneontheentire
and fungi, we focused on the down-regulated genes on the grhl microarray (other than grhl itself) was chitinase 1 (NCU04883)
AHC microarrays. (Figure3),thelackofwhichislikelytocontributetotheconidial-
separationphenotypeobservedingrhl/csp-2mutants.Thechitinase
Highly Enriched FunCat Categories of the Down- 1geneofNeurosporahastwoconsensusGRHLDNAbindingsites
regulated Genes from the Neurospora grhl AHC (AAACGGTT&CACCGGTT)within875bpoftheATGcodon
for the chitinase 1 gene. This suggests that the microarrays had
Microarrays
identifiedatleastsomebiologicallyrelevantgenesthataredirectly
In order to parse microarray results, researchers often use the
regulated by GRHL. There were at least six other GRHL-
Gene Ontology (GO) functional annotation system (www.
dependent down-regulated genes that encode proteins predicted
geneontology.org) to look for highly enriched classes of genes. As
(fromresearchonotherfungi)orknowntobeinvolvedinnormal
a comprehensive GO annotation of the Neurospora crassa genome
‘‘Cell Wall Structure’’ (Figure 3). All of these proteins contain
did notexistat thetimeof theseanalyses,weusedan alternative
predicted secretion signals, and four are experimentally verified
classification system – The Functional Catalogue (FunCat) – for
componentsoftheNeurosporacellwall:gel1(NCU07253,whichhas
which there did exist a high-quality annotation for Neurospora
anAACCGGTTsequence,130bpupstreamofthetranscription
genes [41]. For the 83 genes that were seen to be significantly
start), Mwg1 (NCU05974, which has an AACCGGTT sequence
down-regulated, there were highly significant enrichments in five
,400bpupstreamofthe59endoftheopenreadingframe),non-
FunCat categories (Table 1). Three of these categories are
anchoredcellwallprotein-5(NCU00716,whichhasanAACAGGTT
composed of genes involved in amino acid metabolism –
sequence ,1.8kb upstream of the transcription start), and BYS1
specifically that of cysteine, phenylalanine, and tryptophan. A
domain-containing protein (NCU08907) [46]. Three of the down-
fourth category, ‘‘C-compound and carbohydrate transport’’, is
regulatedcellwallgenes–Mwg1andNCU04431(bothbelonging
composed of membrane transport proteins. The fifth highly
to glycoside hydrolase family 16) as well as gel1 (belonging to
significant category found was ‘‘disease, virulence, and defense’’,
glycosidehydrolasefamily72)–encodebeta-1,3-glucanases.Beta-
which is composed of genes predicted to be involved in fungal
1,3-glucansarethemajorbiopolymerconstituentofthecellwallin
pathogenicity, defense against other organisms, and certain stress filamentous fungi, and it has been shown in many fungal species
responses. thatbeta-1,3-glucanaseenzymesareveryabundantcomponentsof
We could find no direct connections in the literature between thecellwall,wheretheyplayanactiveroleincellwallbiosynthesis
cysteine metabolism and barrier formation in animals. However, andremodeling,aswellasinprocessessuchasbiofilmformation
most amino-acid-metabolism networks are interlinked, and three [46–49]. For instance, mutations in enzymes from glycoside
ofthesegenes(NCU05499,NCU09183,andNCU01402)arealso hydrolase family 72causecell walldefects inS. cerevisiae [48]and
part of the significantly enriched phenylalanine- and tryptophan- alsoaffectmorphogenesisandvirulenceinAspergillusfumigatus[50].
metabolism FunCat categories, for which there are some We also found that seven of the 29 down-regulated genes that
intriguingconnectionstobarrierformationinanimals.Melaniza- could not be assigned a function encode proteins with predicted
tion reactions in Drosophila are used to harden and cross-link secretion signals, and therefore might be components of the cell
cuticular structures, and are known (at least in the epidermis) to wall (Figure 3).
rely on GRH for activation [3]. The reactive quinone molecules We classified 15grhl-dependent down-regulated genes in the
used to carry out these processes are derivatives of dopamine, category ‘‘Virulence/Defense/Detoxification’’ (Figure 3). Defense/
which is itself a derivative of the amino acids tyrosine and VirulencehavenotbeenstudiedexperimentallyinNeurosporacrassa,as
phenylalanine(forareviewsee[42]).Itispossiblethatanancestral itsnormalhost-pathogenrelationshipsareunknown,sothefunctions
roleinphenylalanineregulationbyGRH-liketranscriptionfactors of the following genes are inferred from research in other fungi.
could have been co-opted by cuticle-forming animals for use in Genes potentially involved in defense include the following:
cross-linking apical extracellular barriers. As for the last amino- kynureninase (NCU09183, which has two ACAGGTT sites ,
acid-relatedFunCatcategory,‘‘degradationoftryptophan’’,there 650bp upstream of the open reading frame) and indoleamine 2,3-
is some evidence that it is a general mechanism of all cells to dioxygenase(NCU01402),whichareinvolvedintryptophancatabolism
degrade tryptophan in response to infection, which is used as a andmicrobialgrowthcontrol[43](seeabove);NCU05495encodesa
means to slow microbial growth through tryptophan deprivation putative anti-viral factor [51,52]; and exo-beta-1,3-glucanase
[43]. If true, this function would link tryptophan degradation to (NCU04850) is possibly involved in the degradation of foreign
thefifthFunCatcategory–‘‘disease,virulence,anddefense’’–the polysaccharides. Other Neurospora grhl-dependent genes potentially
presence of which we found especially intriguing, due to the involved in fungal virulence include the following: the metallopro-
numerous documented connections in animals between physical tease MEP1 (NCU07200), whose homolog in C. posadasii has been
epidermal barriers and chemical defense against pathogens shown to be crucial for evasion of host-detection [53]; the p450
[44,45]. monooxygenase lovA (NCU05376), whose homolog in a Fusarium
specieshasbeenshowntobedirectlyinvolvedinmycotoxinsynthesis
Down-regulated Genes from the Neurospora grhl AHC [54]; cerato-platanin (NCU07787), which is potentially important for
Microarrays phytotoxin synthesis [55]; the integral membrane protein pth11
(NCU06328),whosehomologinanotherfungalspeciesisimportant
Toinvestigatethedown-regulatedgenesfromtheNeurosporagrhl
forappressoriumformation[56];andNCU03643,whichencodesa
AHCsamplesinmoredetail,weundertookamanualclassification
cutinase transcription factor that is likely to control plant cuticle
ofthesegenesbasedondatabaseandliteraturesearches.Wewere
digestion during fungal infection [57]. Finally, several genes
especially interested in finding studies carried out directly on the
potentially involved in the detoxification of harmful chemicals and
Neurospora crassa genes or on their close homologs in other fungal
the stress response include the following: the p450 gene pisatin
species. Of the 83 significantly down-regulated genes, 54 had
demethylase(NCU06327),whoseorthologisimportantinfungalpea-
known functions, or predicted functions based on homology to
pathogensfordetoxifyinghostdefensivechemicals[58];thealdehyde
genes inother fungi (Figure3).
dehydrogenase gene (NCU03415), which encodes a broadly acting
PLoSONE | www.plosone.org 6 May2012 | Volume 7 | Issue 5 | e36254
GrainyHead-LikeFunctioninAnimalsandFungi
Table1.Enrichedfunctionalcategoriesforthedown-regulatedNeurosporagrhlgenesandthemisregulatedDrosophilagrhgenes.
Down-regulatedgenesfromNeurosporagrhlAHCsamples
EnrichedFunCatCategories FunCatID #ofgenes p-value
metabolismofthecysteine-aromaticgroup 01.01.09 6 3.07E204
metabolismofphenylalanine 01.01.09.04 3 1.63E203
C-compoundandcarbohydratetransport 20.01.03 5 1.76E203
degradationoftryptophan 01.01.09.06.02 2 3.44E203
disease,virulence,anddefense 32.05 6 4.13E203
MisregulatedgenesfromDrosophilagrhsamples
EnrichedGOBiologicalProcessCategories GOtermID #ofgenes p-value
carbohydratemetabolicprocess 5975 244 1.06E206
chitinmetabolicprocess 6030 77 2.11E206
defenseresponse 6952 117 2.67E206
responsetobioticstimulus 9607 109 2.98E206
aminoglycanmetabolicprocess 6022 95 5.55E206
responsetootherorganism 51707 104 6.35E206
immuneresponse 6955 121 1.57E205
polysaccharidemetabolicprocess 5976 102 2.17E205
humoralimmuneresponse 6959 74 5.40E205
responsetostress 6950 347 8.36E205
EnrichedGOMolecularFunctionCategories GOtermID #ofgenes p-value
structuralconstituentofcuticle 42302 96 1.02E215
structuralconstituentofchitin-basedcuticle 5214 92 2.32E214
serine-typeendopeptidaseactivity 4252 165 7.63E214
serinehydrolaseactivity 17171 185 1.54E213
serine-typepeptidaseactivity 8236 183 1.85E213
peptidaseactivity,actingonL-aminoacidpeptides 70011 352 8.71E209
endopeptidaseactivity 4175 274 1.96E208
structuralconstituentofchitin-basedlarvalcuticle 8010 35 4.81E208
peptidaseactivity 8233 361 1.17E207
chitinbinding 8061 66 1.85E207
polysaccharidebinding 30247 87 4.75E207
(Top)EnrichedFunctionalCatalogue(FunCat)categoriesforthe83significantlydown-regulated(FDR,0.01)genesfromtheNeurosporagrhlAHCmicroarrays.
(MiddleandBottom)ThetopenrichedGeneOntology(GO)‘‘BiologicalProcess’’and‘‘MolecularFunction’’categoriesforallmisregulatedgenesfromtheDrosophila
grhIMembryomicroarrays.
doi:10.1371/journal.pone.0036254.t001
detoxification and metabolic enzyme; the genes catalase-3 Microarray Profiling of Late-stage Drosophila grh
(NCU00355)(anotherverifiedcomponentofthecellwall[46])and Embryos
NAD(P)transhydrogenase(NCU01140),whichencodeenzymesknown
As a comparison to the Neurospora microarray dataset, we also
to be important for oxygen-radical detoxification; and the YBH1
carriedoutmicroarray-based transcriptomeprofilingofDrosophila
flavohemoglobingene(NCU10051),whichmaybeinvolvedinthestress grhIMandcontrolwild-typeembryoscollectedduringlate-stage16
response[59].
andearly-stage17ofembryogenesis[60],whencuticledeposition
Taken together, these results suggest that Neurospora GRHL isoccurring.WeusedflieshomozygousforthegrhIMallelebecause
plays an important role in the regulation of genes that form and
itisthestrongestgrhalleleavailable(withrespecttoitscuticleand
remodelthecellwall(atleastindevelopingconidia).Additionally,
head-skeleton phenotypes) and because homozygous embryos do
a significant number of GRHL-dependent down-regulated genes
not produce any detectable GRH protein (assayed using an
intheaerialhyphaeandconidiaareinvolvedinvirulence,defense,
antibody against the C-terminal half of the protein [6]). By
and detoxification. It is should be noted that the cell wall and
sequencingthegrhIMtranscript,weidentifiedthelesionresponsible
virulence/defense categories are not mutually exclusive, as seven
for the grhIM allele as a TAT to TAA stop-codon introduction in
ofthe15‘‘defensive’’geneproducts(e.g.,cerato-plataninandMEP1)
exon seven, in the N-terminal end of the DNA-binding domain
havesecretionsignals(Figure3)andarelikelytobedepositedinto
and about half-way through the protein (Figure 1B, amino acid
thecell wallor released intotheextracellular space.
PLoSONE | www.plosone.org 7 May2012 | Volume 7 | Issue 5 | e36254
GrainyHead-LikeFunctioninAnimalsandFungi
Figure3.Down-regulatedgenesfromtheNeurosporagrhlAerialHyphaeandConidiamicroarraysamples.Amanualclassificationofthe
significantlydown-regulatedgenesfromtheNeurosporagrhlAHCmicroarrays.‘‘BroadID’’entriescorrespondtothegeneIDsfoundintheBroad
InstituteNeurosporacrassadatabase.ThetwoitalicizedentriesinthiscolumnrefertoprobesthatdonotcorrespondtogenesintheBroaddatabase,
PLoSONE | www.plosone.org 8 May2012 | Volume 7 | Issue 5 | e36254
GrainyHead-LikeFunctioninAnimalsandFungi
butwhichcorrespondtogenesintheMIPSdatabase.‘‘GenenameorDescription’’and‘‘Function’’entrieswerebasedontheannotations
foundintheBroadandMIPSdatabases,aswellliteratureandhomologysearches.Numbersincurlybracketsindicategenesthatbelongtooneofthe
five highly enriched FunCat categories: {1} metabolism of the cysteine - aromatic group, {2} metabolism of phenylalanine, {3} C-compound and
carbohydratetransport,{4}degradationoftryptophan,and{5}disease,virulenceanddefense.Entrieswithasterisksencodeexperimentallyverified
componentsoftheNeurosporacellwall[46].‘‘Fold(wtvalue)’’entriesindicatethefoldchangesobservedingrhlmutantaerialhyphaeandconidia
(relativetowildtype);wild-typemicroarrayfluorescencevaluesareshowninparentheses(thebackgroundlevelwas,100units).‘‘FDR’’entries
indicatetheFalseDiscoveryRatevaluescalculatedforeachgene;onlygeneswithFDRvalueslessthan0.01areshown.Columns1–9ofthegrid
representasimplificationoftheFunCatclassificationsystem;solid-coloredblocksindicatethosegenesareclassifiedinthecorrespondingFunCat
categories;dashesindicatethatwefoundevidenceintheliteraturetosuggestthesegenesbelonginthecorrespondingcategories.Column10of
thegridindicateswhethertheencodedproteinsarepredictedtobesecreted,accordingtotheSignalP(S)orTargetP(T)predictionalgorithms.
Significantlydown-regulatedgenesthatcouldnotbeassignedafunctionarenotshowninthisfigure.
doi:10.1371/journal.pone.0036254.g003
Y29 in the D.mel GRH DNA-binding domain; see Figure S4 for initially puzzling, as most of these genes were seen to be up-
details); this mutation is consistent with a functional null regulatedinthegrhIMmutants(Table2),andGRHhasnoknown
phenotype for grhIM and the lack of GRH protein detection with functionas an inhibitorof theimmuneresponse.
aC-terminalspecificantibody[6].SeetheMaterialsandMethods From these we results, we conclude that in addition to the
and Text S1 for more details on the Drosophila microarrays and expectedmisregulationofgenesinvolvedincuticleformation,late-
dataanalyses.Besidesthemutationingrh,thecontrolandmutant stage grhIM embryos are experiencing a massive wound/immune
embryosdifferedslightlywithrespecttotheirgeneticbackgrounds, responseaswell.Duringthestagestheywerecollected(late-stage
as the wild-type strain contained the yellow1 allele, which has an 16 or early-stage 17 of embryogenesis) the grhIM embryos have
adult pigmentation defect, while the grhIM embryos were yellow+. weaker and more permeable epidermal barriers [1,6,12], yet are
However,wedonotbelievethissignificantlyinfluencedourresults still motile, which can cause their fragile cuticles to rupture.
since our microarray data indicate that yellow is expressed at Consistent with this, the pale gene, which encodes tyrosine
extremely low levels during the embryonic stages we tested; hydroxylase, is known to be up-regulated around sterile wound
furthermore, the yellow transcript expression levels were not sites in a largely grh-independent manner [3,4], and in grhIM
significantly different between the grhIM and control embryos on embryos,paletranscriptsaresignificantlyup-regulated.Thisisalso
themicroarrays. consistent with the observation that clean puncture wounding of
late-stage embryos (in the absence of intentional microbial
Highly Enriched Gene Ontology Categories of the infection)alsoinducestheexpressionoflargenumbersofDrosophila
Misregulated Genes from the Drosophila grh Embryo genes involved in innate immunity and the stress response (R.
Microarrays Patterson &W.McGinnis,unpublished).
Assayedatstages16–17ofembryogenesis,zygoticlossofGRH
function has a huge impact on the Drosophila transcriptome as a Misregulated Genes from the Drosophila grh Embryo
whole, as over 1,200 genes (FDR ,0.01) were seen to be Microarrays Reflect the Role of GRH in Barrier Formation
misregulated (up or down) in grhIM mutants compared with wild and Wound Healing
type (see the Materials and Methods section for the accession
Wecarriedoutamanualclassificationofthegenesbothandup-
numbers of the Drosophila microarray datasets). Verification of and down-regulated on the Drosophila grhIM embryo microarrays,
microarray fold-change directionality for eight genes using
andselectgenesareshowninTable2.Weplaced64genesinthe
quantitative RT-PCR isshownin FigureS5.
category ‘‘Cuticle Formation/Chitin Metabolism’’, including
A search for enriched GO ‘‘Biological Process’’ (BP) and
genes involved in the generation and degradation of chitin
‘‘Molecular Function’’ (MF) categories was performed (Text S1),
molecules, as well as genes for many cuticle proteins that are
and the top ten and eleven most significant classes, respectively,
deposited into the cuticle to mediate aspects of cuticle-shape and
are shown in Table 1 (see Table S1 for the full lists of the
elasticity [61,62]. The majority of these genes (42 of 64) were
significantly enriched GO-BP, GO-MF, and GO ‘‘Cellular
down-regulated, consistent with potential direct regulation by
Component’’categories).AsGRHisknowntobeveryimportant
GRH,andconsistentwiththeideathatGRHisacrucialregulator
for cuticle development and wound healing in Drosophila, we
of physical-barrier formation in Drosophila. However, a subset of
expected to see numerous genes involved in these processes
these"cuticle/chitin"genes(22of64)wereseentobeup-regulated
misregulated on the microarrays. Indeed, four of the most
in the grh mutants. It is possible that these up-regulated cuticle
significant GO-BP classes (e.g., ‘‘chitin metabolic process’’ and
genes are normally directly repressed by GRH in wild-type
‘‘aminoglycanmetabolicprocess’’)andfiveofthemostsignificant
embryos, but we believe it more likely that they are being
GO-MF classes (e.g.,‘‘structural constituent of the cuticle’’ and
overexpressed to compensate for the lack of the GRH-activated
‘‘chitin binding’’) are consistent with the known role of GRH in
cuticle proteins, or they are being overexpressed in response to
regulating theformation of chitin-basedcuticular barriers.
cuticular damagein grhIMmutants (see above).
Surprisingly, the remaining six GO-BP categories (of the top
Interestingly,oneofthemoststronglydown-regulatedgeneson
ten)wereallcomposedofgenesinvolvedineitherinnateimmunity
themicroarraywaschitinase3(,13folddown)(Table2),whichisa
or the stress response (e.g., ‘‘defense response’’, ‘‘immune
Drosophila homolog of Neurospora chitinase 1– the most strongly
response’’, and ‘‘humoral immune response’’). Similarly, the
down-regulatedgeneintheNeurosporagrhlmutants(seeabove).We
remaining six GO-MF categories (of the top eleven) were all
identifiedthreehigh-affinityGRHbindingsiteswithinthetwokb
composed of genes that encode products with either serine-
protease orserine-protease-inhibitor activity.This was interesting region upstream of the Drosophila chitinase 3 transcriptional start
because serine protease cascades are used to trigger the site, and chitinase 3 is also extensively co-expressed with GRH
hemolymph melanization-reactions used in response to infection, throughout the Drosophila epidermis and tracheal system during
and serine protease inhibitors (also known as serpins) are used to embryogenesis (data not shown), consistent with direct regulation
limit the spread of this reaction (for a review see [44]). This was by GRH.
PLoSONE | www.plosone.org 9 May2012 | Volume 7 | Issue 5 | e36254
GrainyHead-LikeFunctioninAnimalsandFungi
Table2. Select misregulatedgenes from thelate-stage grhIM embryo microarrays.
CuticleFormation/ChitinMetabolism(64)
CG# GeneNameorSymbol ProteinType/Process Fold(wtvalue)* FDR
CG2044 Lcp4 cuticleprotein 2139.41(13854)* 2.36E207
CG30163 Cpr60D cuticleprotein 251.83(5170)* 1.20E206
CG18066 Cpr57A cuticleprotein 232.87(38041) 4.27E206
CG15515 2 cuticleprotein 228.37(75931) 7.15E206
CG2043 Lcp3 cuticleprotein 214.34(1067)* 3.64E205
CG18140 Chitinase3 chitinmetabolism 212.9(2331) 2.48E205
CG7941 Cpr67Fa1 cuticleprotein 211(109921) 4.05E205
CG6955 Lcp65Ad cuticleprotein 29.9(1824) 5.80E205
CG4052 Cpr5C cuticleprotein 28.72(4115) 7.31E205
CG8697 Lcp2 cuticleprotein 26.65(422)* 1.60E204
CG32400 Lcp65Ab1 cuticleprotein 26.64(73278) 1.65E204
CG17052 obstructor-A cuticleorganization 25.67(106803) 2.64E204
CG8510 Cpr49Af cuticleprotein 25.66(5870) 2.75E204
CG9070 Cpr47Eg cuticleprotein 25.54(11869) 2.79E204
CG6217 knickkopf cuticleorganization 25.29(5171) 3.19E204
CG14250 TweedleQ cuticleprotein/bodyshape 25.09(533) 3.72E204
CG7287 Lcp65Aa cuticleprotein 24.94(1261) 3.91E204
CG4778 obstructor-B cuticleorganization 24.87(16760) 3.88E204
CG18773 Lcp65Ab2 cuticleprotein 24.45(15581) 5.24E204
CG7216 Acp1 cuticleprotein 24.43(3406) 5.02E204
CG14643 TweedleG cuticleprotein/bodyshape 24.02(117093) 6.66E204
CG9369 miniature cuticleorganization 23.77(696) 1.33E203
CG14639 TweedleF cuticleprotein/bodyshape 23.32(30236) 1.79E203
CG10297 Acp65Aa cuticleprotein 23.28(4421) 1.38E203
CG11650 Lcp1 cuticleprotein 23.23(235)* 1.18E203
CG10529 Lcp65Ae cuticleprotein 23.17(12315) 1.31E203
CG5883 2 chitinmetabolism 23.06(2218) 1.38E203
CG7548 2 cuticleprotein 22.75(8307) 2.01E203
CG9535 mummy chitinbiosynthesis 22.63(43714) 2.37E203
CG11142 obstructor-E cuticleorganization 22.56(6997) 2.77E203
CG5494 Cpr92F cuticleprotein 22.5(39488) 2.92E203
CG33302 Cpr31A cuticleprotein 22.5(142683) 2.95E203
CG18779 Lcp65Ag3 cuticleprotein 22.46(233203) 3.08E203
CG12009 2 chitinmetabolism 22.4(2092) 6.86E203
CG7252 2 chitinmetabolism 22.24(3566) 4.48E203
CG9295 Cpr76Bc cuticleprotein 22.2(303) 4.82E203
CG12755 l(3)mbn cuticleprotein 22.18(380) 4.95E203
CG32499 Cda4 chitinmetabolism 22.13(24891) 6.23E203
CG15008 Cpr64Ac cuticleprotein 22.09(2006) 5.95E203
CG18778 Cpr65Au cuticleprotein 22.07(534) 6.47E203
CG32404 Cpr65Aw cuticleprotein 22.03(211) 6.89E203
CG5812 TweedleT chitinmetabolism 21.91(27604) 9.30E203
CG9781 obstructor-G cuticleprotein/bodyshape 1.78(89)* 9.48E203
CG9307 Chitinase5 cuticleorganization 1.78(15030) 8.48E203
CG9079 Cpr47Ea chitinmetabolism 1.84(4076) 8.29E203
CG8515 Cpr49Ah cuticleprotein 1.85(78)* 6.72E203
CG2555 Cpr11B cuticleprotein 1.9(1069) 5.96E203
CG6773 sec13 cuticleprotein 1.95(1358) 4.89E203
CG9665 Cpr73D cuticleorganization 1.99(1041) 6.30E203
PLoSONE | www.plosone.org 10 May2012 | Volume 7 | Issue 5 | e36254