Extensive chordate and annelid macrosynteny reveals ancestral homeobox gene organisation

31 Pages·2011·1 MB·English

by  David Ferrier

Extensive Chordate and Annelid Macrosynteny Reveals
Ancestral Homeobox Gene Organization
Jerome H. L. Hui,(cid:1),1 Carmel McDougall,(cid:2),1 Ana S. Monteiro,§,1 Peter W. H. Holland,1 Detlev Arendt,2
Guillaume Balavoine,3 and David E. K. Ferrier*jj,1
1Department of Zoology, University of Oxford, Oxford, United Kingdom
2EMBL Heidelberg, Heidelberg, Germany
3Institut Jacques Monod, CNRS/University Paris Diderot, France
(cid:1)Present address: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
(cid:2)Present address: School of Biological Sciences, University of Queensland, Brisbane, Australia.
§Present address: Centro Andaluz de Biologia del Desarrollo, Universidad Pablo de Olavide, Seville, Spain.
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jjPresent address: The Scottish Oceans Institute, University of St Andrews, St Andrews, United Kingdom. o
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*Corresponding author: E-mail: [email protected]. Rnlo
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Associate editor: Patricia Beldade d
se
Genbank accession number: PduMox (HQ263150). ead fro
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Abstract rch http
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Genes with the homeobox motif are crucial in developmental biology and widely implicated in the evolution of a://ac
development.TheAntennapedia(ANTP)-classisoneofthetwomajorclassesofanimalhomeoboxgenes,andincludesthe rtade
Hox genes, renowned for their role in patterning the anterior–posterior axis of animals. The origin and evolution of the icmic
ANTP-class genes are a matter of some debate. A principal guiding hypothesis has been the existence of an ancient gene le.ou
p
Mega-cluster deep in animal ancestry. This hypothesis was largely established from linkage data from chordates, and the .c
o
Mega-cluster hypothesis remains to be seriously tested in protostomes. We have thus mapped ANTP-class homeobox m
/m
genes to the chromosome level in a lophotrochozoan protostome. Our comparison of gene organization in Platynereis b
e
dumeriliiandchordatesindicatesthattheMega-cluster,ifitdidexist,hadalreadybeenbrokenupontofourchromosomes /a
by the time of the protostome–deuterostome ancestor (PDA). These results not only elucidate an aspect of the genome rtic
le
organizationofthePDAbutalsorevealhighlevelsofmacrosyntenybetweenP.dumeriliiandchordates.Thisimpliesavery -a
b
low rate of interchromosomal genome rearrangement in the lineages leading to P. dumerilii and the chordate ancestor stra
since the time of the PDA. c
t/2
9
Key words: Platynereis dumerilii, Antennapedia-class, homeobox, protostome–deuterostome ancestor. /1
/1
5
7
/1
Introduction resideindifferentlineages,suchthatcomparisonofthedif- 7
4
8
ferentevolutionaryrelicsoftheMega-clustershouldallow 4
TheMega-clusterhypothesisprovidestheprincipalcurrent 5
9
frameworkforunderstandingtheoriginandearlyevolution its composition to be deduced. by
Alternatives to the Mega-cluster hypothesis can be en- g
of the homeobox-containing genes of the Antennapedia- u
visaged that correspond to either of two general forms. e
class (ANTP-class) in animals. The ANTP-class contains First, at least some nontandem duplications could have st o
such important developmental control genes as the n
beeninvolved,eitherofgenesorofgroupsofgenes,before 2
Hox, ParaHox, and NK genes, widely involved in multiple allthecomponentgenefamiliesofthehypothesizedMega- 1 N
developmental processes across the animal kingdom. o
v
clusterhadarisen,oraprecursorclusterpriortotheMega- e
TheMega-clusterhypothesisproposesthatdeepinanimal m
ancestry, a precursor gene of the ANTP-class (the Proto- cluster may have split apart, such that the complete be
Mega-cluster never in fact existed. This type of scenario r 2
ANTPgene)underwentseveralroundsoftandemduplica- 0
1
tionstoformaclusterofgenesthatweretheevolutionary can be considered as a ‘‘weak’’ form of the Mega-cluster 8
precursors for the genes of the Hox cluster, the ParaHox hypothesissinceeventhoughthecompleteMega-cluster
cluster,theNKcluster,andseveralcloselyrelatedgenefam- did not exist under such a scenario, significant portions
ilies (Pollard and Holland 2000; Garcia-Fernandez 2005) of the ANTP-class still evolved within clustered arrange-
(fig.1).ThisMega-clusterthenbrokeapartinseveralloca- ments. The second alternative is that ANTP-class genes
tions as lineages diverged from this ancestral state, at lo- secondarilycametobeclusteredindistinctanimallineages
cations hypothesized to have been effectively random after originating in locations scattered around ancestral
acrossdifferentanimallineages,apartfrominfunctionally genomes. These different scenarios would be expected
constrained clusters such as the Hox cluster. Different ex- toleadtodifferentANTP-classlinkagesindistinctlineages.
tents of Mega-cluster remains are therefore predicted to The ‘‘strong’’ and weak forms of the Mega-cluster
©TheAuthor2011.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.Allrightsreserved.Forpermissions,please
e-mail:[email protected]
Mol. Biol. Evol. 29(1):157–165. 2012 doi:10.1093/molbev/msr175 Advance Access publication July 4, 2011 157
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Hui et al. · doi:10.1093/molbev/msr175
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tohavethenbrokeninseveralplaces,presumablyindependentlyondifferentanimallineages.Thediagonallinesshowthebreaksdeducedto h
bepresent inthe chordate ancestor, which separated thesegenes intothreedistinct chromosomes. Precise geneorderisnot known inthe ttp
s
ancestralMega-clusterexceptwithintheParaHox,Hox,andNKclusters.Conventionally,theseANTP-classgenesareclassifiedasParaHox(pale ://a
grayboxes),Hox-like(darkgrayboxes),andNK-like(blackboxes),withDlxbeinganNK-likegene(black)linkedwithHox-likegenes.However, c
a
thisnomenclaturemaybemisleadingandunjustified(seetextfordetails)andweinsteadproposetheuseof‘‘Hox-linked’’and‘‘NK-linked’’,in d
e
which case Dlx is Hox-linked (dark gray) in chordates. Note the relative timing of the duplications to generate the full complement of m
ic
ProtoHox,HoxL,NK,andNKLgenesisstilldebatedandsomecomponentsmaywellhavearisenbeforeothers(e.g.,NKexpansionbeforeHox; .o
u
Larrouxetal. 2007). p
.c
o
m
/m
hypothesis would be expected to lead to similar, but not ternative protostome for comparison, the polychaete
b
e
identical, sets of linked ANTP-class genes in distinct line- Platynereis dumerilii, which resides in the lophotrochozoan /a
ages if the cluster breaks in effectively random locations. superphylum rather than in the Ecdysozoa. P. dumerilii has rtic
le
Distinguishing between the strong and weak forms of ahaploidnumberof14(Jhaetal.1995),achromosomenum- -a
b
the Mega-cluster hypothesis requires determination of ber potentially similar to that of the protostome–deutero- s
tra
ANTP-classlinkagepatternsacrossadiversityofanimallin- stome ancestor (PDA), if the eumetazoan ancestor had c
eages.Incontrast,iftheANTP-classlinkagesevolvedfrom about13linkagegroups(Putnametal.2007)andthechordate t/29
/1
a secondary ‘‘coming together’’ from ancestral scattered ancestorabout17(Putnametal.2008).Combinedwithage- /1
locations, then we would expect a largely random assort- nomesizeofapproximately1(cid:1)109bp,thisreducesthelikeli- 57
/1
mentofANTP-classlinkagesacrossdistinctanimallineages. hood of chance secondary associations of gene linkage that 7
4
8
To date, a comprehensive analysis of the evolution of the have confounded the insect and nematode comparisons. 4
5
Mega-cluster has beenhampered by twomajor problems. The secondproblemisthe difficulty in obtaining robust 9 b
y
Thefirstproblemisthepaucityofchromosome-levelmap- phylogeneticinformationfromtheANTP-classgenes.Dlxis g
u
pingdata. Such data areavailablefrom chordates, whichan- a key gene in the Mega-cluster hypothesis because Dlx is e
s
cestrally, and in the prototypical chordate genome of conventionally deemed to have greater sequence similarity t o
n
amphioxus(Branchiostomafloridae),had/havetheHoxcluster tothegenesoftheNKclusterthantothoseoftheHoxclus- 2
1
andtheANTP-classfamiliesEvx,Mox,Gbx,En,Mnx,andDlx ter, such that Dlx is often referred to as an ‘‘NK-like’’ gene N
o
ononechromosome,theParaHoxclusteronasecondchro- rather than a ‘‘Hox-like’’ gene. Since this supposed NK-like ve
m
mosome,andtheremainsoftheNKclusteronathirdchro- gene is linked to the Hox cluster in chordates, it has been b
e
mosome (Castro and Holland 2003; Luke et al. 2003; Castro held toprovideevidencefor the ancient linkageof NK-like r 2
0
etal.2006).Toextendthedepthofanimalancestrytowhich and Hox-like genes in the Mega-cluster, with an interchro- 1
8
the extent of the hypothesized Mega-cluster can be traced, mosomal translocation occurring that separated all of the
comparisonstoprotostomesarerequired.Chromosome-scale NK-like genes, except for Dlx, from the Hox-like genes
resolutionin protostomesiscurrentlyrestrictedtotheecdy- (Pollard and Holland 2000; Garcia-Fernandez 2005)
sozoansuperphylum(Coulieretal.2000;CastroandHolland (fig. 1). The lack of robust resolution of many interfamily
2003). Unfortunately, the low chromosome number in such relationships within the ANTP-class in molecular phyloge-
ecdysozoanmodelsasDrosophilaandCaenorhabditiselegans niesbasedonhomeodomainsequences,particularlythere-
meansthatthesesystemsprovidelittleresolution,duetothe lationship of the Dlx family to the Hox and NK genes, has
difficulty in distinguishing ancestral linkage patterns from importantimplicationsfortheMega-clusterhypothesis.No-
chance secondary associations. Also, a high level of genome tably, Dlx is not robustly classified as being more similar
rearrangementhasbeendescribedforinsectsandnematodes to Hox or NK genes via molecular phylogenies; in different
(Zdobnov et al. 2005; Zdobnov and Bork 2007), further con- analyses,DlxhasbeenplacedclosertotheNKclustergenes,
foundingelucidationofancestrallinkages.Here,weuseanal- the Hox cluster genes, or even outside both NK and Hox
158
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Annelid to Chordate Homeobox Macrosynteny · doi:10.1093/molbev/msr175
genes together, and never with strong support values 5#-TTCCATTTCATTC(G/T)TC(T/G)GTTTTG-3# were desig-
(Howard-Ashby et al. 2006; Monteiro et al. 2006; Ryan ned based on sequences from the Mox genes of other
etal.2006;Larrouxetal.2007;Takatorietal.2008;reviewed bilaterians(supplementaryfig.S1,SupplementaryMaterial
in Ferrier 2008). Furthermore, in an attempt to discover online).NewPdMoxF2wasusedasthenestedprimerafter
diagnostic residues for both the so-called Hox-like and theprimaryPCRofNewPdMoxF1andNewPdMoxRP.PCR
theNK-likegroups,theDlxfamilycouldnotbeaccommo- wasperformedonthegenomicDNAofP.dumerilii,andthe
dated(Fonsecaetal.2008).Thisemphasizestheweaknessof parameterswere94(cid:1)Cfor5min,35times(94(cid:1)Cfor1min,
the Hox-like and NK-like superfamily terms. Chromosome 55 (cid:1)C for 1 min, and 72 (cid:1)C for 1.5 min), and 72 (cid:1)C for 10
location and linkage patterns are perhaps a more robust min.Cloningandsequencingwereperformedasdescribed
guidetoancestralassociationsbetweenANTP-classfamilies; above. 3#RACE was carried out following the manufac-
hence,ourproposalthatthetermsHox-likeandNK-likebe turer’s instructions (SMART RACE cDNA Amplification
abandoned and ‘‘Hox-linked’’ and ‘‘NK-linked’’ adopted as Kit, Clontech). Gene-specific primer PdMox3#RACE-F1,
D
more informative alternatives (Ferrier 2008). 5#-CCATACACAACTACCTGACGAGACTGAG-3#,wasde- ow
The key question is then, which genes are linked to the signed based on the sequence information obtained from nlo
HoxandNKclustersanddobilaterianlineagesexhibitover- PCR with degenerate primers. PCR was performed on 3#- ad
e
limapppliynganpaintttearcntsMoefgHao-cxlu-satnerdiNnKth-leinkPeDdAgeanneds?inTdheipsewndoeunldt RunAiCveEr-sRaelapdyrimcDeNr,AUuPsMing(5t#h-eCgTeAnAe-TsApeCcGifiAcCpTrCimAeCrTaAnTdAtGhe- d from
breakups along diverging lineages. The alternative scenario, GGCAAGCAGTGGTATCAACGCAGAGT-3#). Five cycles http
oilifemsinatdchivienrgsepbatiltaetrenrsiaonfliHnoexa-gleins,kwedoualnddinNstKe-alidnkimedplgyetnheatfatmhe- f(o9r43(cid:1)0Cs,fo7r03(cid:1)C0 fsoarn3d0s7,2an(cid:1)dC7f0or(cid:1)C3fmorin3),mfiivne),cayncdle2s5(9cy4cl(cid:1)eCs s://ac
a
Mega-clusterhadalreadybrokenuptothisextentbythetime (94 (cid:1)C for 30 s, 68 (cid:1)C for 30 s, and 70 (cid:1)C for 3 min) of de
m
ofthePDAorthatanextensiveMega-clusterdidnotinfact amplification were carried out. The PCR products were ic
exist. Here, we test this by determining the locations of the analyzedona1%agarosegel.PurificationofDNA,ligation, .ou
p
ANTP-class genes to discover whether the patterns of Hox- transformation, and sequencing were performed as de- .c
o
linked and NK-linked genes in chordates and a protostome scribed above. Sequences were aligned with other Mox m
/m
like Platynereis overlap or instead show the same divisions. genes as shown in supplementary fig. S1, Supplementary b
e
We have mapped ANTP-class homeobox genes to the Material online. The PduMox cDNA is deposited in /a
chromosome level in a lophotrochozoan protostome, Genbank with accession number HQ263150. rticle
the annelid P. dumerilii, for the first time and compared -a
b
BAC Library Screening s
this gene organization in P. dumerilii with available tra
wMheoglae--cgleunstoemr,eif isteqduidenecxeisst., hOaudraldreaatadyrbervoekaelnthuaptonthtoe GcloennoesmwicerbeacistoerlaiatledaratisficdieaslccrhibreodmionsoHmuei e(tBAalC. )(2a0n0d9)puhsaingge ct/29/1
four chromosomes by the time of the PDA. These results cDNA fragments as probes (details in supplementary /1
5
material, Supplementary Material online). Probescontaining 7
notonlyelucidateanaspectofthegenomeorganizationof /1
the ANTP-class homeobox genes were generated with the 7
the PDA but also reveal high levels of macrosynteny be- 4
8
PCR DIG labelling mix kit (Roche, Germany). DNA was pu- 4
tween P. dumerilii and chordates. This implies a very 5
low rate of interchromosomal genome rearrangement in rifiedwiththeGFXPCRDNAandGelBandPurificationKit 9 b
y
the lineage leading to P. dumerilii since the time of the (Amersham Biosciences). Filters containing the BAC library gu
constructed for P. dumerilii (BACPAC Resources, Children’s e
PDA,makingthisannelidanexcellentcandidatefortracing s
Hospital Oakland Research Institute, http:// t o
the genome-scale evolution of bilaterian genomes from n
bacpac.chori.org/library.php?id5209) were screened with 2
their PDA origins, over 550 Ma. 1
DIG-labeled probes according to the DIG nucleic acid N
o
Materials and Methods detection kit (Roche, Germany). Hybridization was carried ve
m
out at 42 (cid:1)C overnight with gene-specific probe (25 ng/ml b
e
Gene Cloning DIGEasyHybbuffer)andwashed(2(cid:1) salinesodiumcitrate r 2
0
Productsfrompolymerasechainreactions(PCRs)werere- [SSC]/0.1%sodiumdodecylsulfate[SDS],roomtemperature, 1
8
solvedby1%agarosegelelectrophoresisandthebandsof and2(cid:1)15min;0.5(cid:1)SSC/0.1%SDS,60(cid:1)C,and2(cid:1)15min).
appropriatesizespurifiedwiththeGFXPCRDNAandGel HybridizationsignalsweredetectedbyCDP-star,ready-to-use
BandPurificationKit(AmershamBiosciences).Thepurified (Roche,Germany),andexposedtoBioMaxLightfilm(Kodak).
DNAwasligatedwiththepGEM-TEasyVector(Promega)
and transformed into XL10-blue chemically competent
cells. The QIAprep Spin Miniprep Kit (Qiagen) was used BAC Clone Preparation and Sequencing
to purify the plasmids, and sequencing of plasmids was EachBACclonewaspurifiedwiththeQIAfilterPlasmidMidi
done with BigDye Terminator v3.1 cycle sequencing kit kit (Qiagen) and prepared for chromosomal fluorescent in
(Applied Biosystems). situ hybridization (FISH) afterconfirming the gene content
DegenerateprimersNewPdMoxF15#-AAACCAAGGAAG- by restriction enzyme digestion (EcoRI and HindIII as fre-
GA(A/G)AG(A/G)AC(A/G)GC-3#, NewPdMoxF2 5#-ACCA- quent cutters, NotI to check the insert size by cutting
AACA(C/T)CAGATCC(A/G)(T/G)GAA, and NewPdMoxRP out the insert with restriction sites flanking the polylinker
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Hui et al. · doi:10.1093/molbev/msr175
promoter regions of the vector), gene-specific primer PCR, Results
andsequencing.TodiscoverwhethersomeBACcloneswere
Dlx Is Hox-Linked Rather than NK-Like
overlappingandtodesignprimersforgenomewalkingatthe
InP.dumerilii,wefindthatPduDlxislinkedtotheHoxcluster,
endsofBACclones,someBACclonesweresubjectedtoend
resemblingthesituationinchordates.ABACclonecontain-
sequencing.Stepswerecarriedoutasdescribedinthepro-
ingPduDlxlocalizestothetelomericendofachromosometo
tocol of the BigDye Terminator v3.1 cycle sequencing kit
whichBACclonescontainingHoxclustergenesalsolocalize,
(Applied Biosystems) with primers pTARBAC2.1T7,
5#-GCCGCTAATACGACTCACTATAGGGAGAG-3#, and withtheHoxprobeslabelingasiteclosertothecentromere
pTARBAC2.1Sp6, 5#-ACTGTGGCTTGTTTTACAATTT-3#, (e.g.,PduLox5,fig.2e),exceptfortheHoxgenePduPost1(see
below).Giventheambiguityoftheinterfamilyrelationships
with the following modifications. Each reaction consisted
betweentheANTP-classhomeoboxgenes,particularlythat
of10pmolsequencingprimer,2.5ll5(cid:1)BigDyesequencing
ofDlxrelativetotheHoxandNKclustergenefamiliesinmo-
buffer, 2 ll ready reaction premix, and 0.5–1 lg of DNA D
lecularphylogenies(seeIntroduction),wehererefertoDlxas o
templateandwaspreparedandrunthroughthermalcycles w
aHox-linkedgeneinbothPlatynereisandchordatesrather n
of96(cid:1)Cfor1minand50cycles(96(cid:1)Cfor30s,50(cid:1)Cfor10s, lo
thanusethepossiblymisleadingtermNK-like. a
and 60 (cid:1)C for 4 min). de
d
Absence of Hox and ParaHox ‘‘Cluster’’ Linkage fro
Genomic Phage Library Screening m
TheHoxandParaHoxclusterswerepostulatedtohaveorig- h
Since some of the screenings of the BAC library did not inatedfromtheduplicationofasingleancestralclusterthat ttps
provide any positive signals with several probes, including existedearlyinmetazoanevolution,theProtoHoxgeneclus- ://a
PduEmx, PduGbx, PduEngrailed, PduVax, PduNot, PduLbx, c
ter(Brookeetal.1998).Itisunclearwhetherthisseparation ad
andPduNK5probes,agenomicphagelibraryofP.dumerilii, e
into Hox and ParaHox clusters was a tandem duplication m
synthesized by Lofstrand LabsLtd., was screened. Prepara- ic
(MinguillonandGarcia-Fernandez2003),atrans-duplication .o
tion of the host strains, phage infection, plating cultures, u
andplaqueliftswereallcarriedoutasinthemanufacturer’s (Brooke et al.1998), or a break of a single long clusterinto p.co
two(Ryanetal.2007).InP.dumerilii,theHoxlocus(intact m
instructions (Lambda FIX II Library, Stratagene). The pri- apart from PduPost1, see below; Monteiro AS and Ferrier /m
mary library contained 5 (cid:1) 105 pfu/0.4 ml of SM buffer DEK, unpublished) and the ParaHox locus (split in two be/a
(100 mM NaCl, 8 mM MgSO4.7H2O, 50 mM Tris–HCl, but on a single chromosome; Hui et al. 2009) are located rtic
0.002% gelatin) with 7% dimethylsulfoxide (DMSO); le
whereas the amplified library contained 2.6 (cid:1) 1010 pfu/ on two different chromosomes.PduCdx, whichisrepresen- -ab
tativeoftheParaHoxchromosome(fig.2o),labelsachromo- s
ml of SM buffer with 7% DMSO. The host bacteria strain some that is distinct from the Hox cluster-bearing trac
wasXL-1BlueMRA(P2).Theaverageinsertsizecontained chromosomelabeledbyPduLox5(fig.2s).Thisisthefirstex- t/29
within the vector is about 17 kb. Two amplified phage li- ample of Hox and ParaHox clusters being mapped to two /1/1
braryplateswerescreenedat;200,000pfuoneachplate. 5
different chromosomes in a lophotrochozoan protostome. 7
Plaquesweretransferredontoachargednylonmembrane /1
7
4
(Hybond Nþ, Amersham Biosciences) with hybridization, Hox-Linked Genes and PduPost1 84
washing, and detection steps carried out as for the BAC In the formulation of the Mega-cluster hypothesis, the 59 b
library screening described above. Secondary and tertiary chordate Hox chromosome contains several other y g
screenings were further performed before a single plaque u
ANTP-class genes in addition to the Hox genes and Dlx. e
s
was isolated for large-scale DNA preparation with Wizard These include three genes originally called the EHGbox t o
n
DNA Clean-up Systems (Promega). All phages were sub- genes(Engrailed[en],HB9/Mnx,andGbx),plusMox/Meox 2
1
jectedtogene-specificprimersequencingbeforeperform- and Evx; this last gene having been a member of the an- N
o
ing chromosomal FISH. A phage containing PduLbx and cestral Hox cluster and retained in this location in ve
m
a phage containing PduNK5 were confirmed. chordates and cnidarians (Miller and Miles 1993; Pollard b
e
and Holland 2000; Garcia-Fernandez 2005; Ryan et al. r 2
Chromosomal FISH 2007; Hollandet al.2008). Here, we showthat P. dumerilii 01
8
Two-color FISH was performed as described in Hui et al. orthologues of Mnx, Mox, and Evx are still Hox-linked
(2009). Briefly, genomic clones were labeled with either (fig. 2a, b, and d). PduEvx, despite still being Hox-linked
digoxigenin or fluorescein-12-dUTP and nick translation in Platynereis, is however no longer a member of the
and detected with DIG-rhodamine Fab fragments plus Platynereis Hox cluster, with PduPost2 (and the rest of
Texas-Red anti-sheep antibody or Alexa Fluor 488 signal- the Hox cluster) being relatively close to the centromere,
amplification kit, respectively. Pairs of ANTP-class probes whereasPduEvxislocatedclosertoatelomere(fig.2b).This
were hybridized to metaphase chromosome spreads, which disruption of the ‘‘posterior’’ end of the Hox cluster is
werecounterstainedwith4’,6-diamidino-2-phenylindole.Im- further demonstrated by loss of the posterior Hox gene
ageswerecapturedwithaZeissAxioskopmicroscopeequip- PduPost1 from the cluster (fig. 2). Here, we show that
ped with an Axiocam camera. All images were processed notonlyhasPduPost1beenrelocatedoutoftheHoxcluster
with whole-layer color adjustment on the complete image (fig. 2c) but also that it has been translocated to the ‘‘NK
with Adobe Photoshop 7.0. cluster’’ chromosome (discussed below) (fig. 2h and k).
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FIG. 2. Chromosomal FISH mapping the positions of pairs of ANTP-class homeobox genes in the protostome Platynereis dumerilii. All the
localizedP.dumeriliiANTP-classhomeoboxgenesarelocatedononlyfourchromosomes,representedbycoloredboxes:red,Hoxchromosome
(a–e);green,NKchromosome(f–l);orange,NK2chromosome(m–n);purple,ParaHoxchromosome(o).Thepalebluebox(p–v)represents
the gene pairs that distinguish the four Platynereis ANTP-class chromosomes from each other. Scale bars 5 20 lm. For further details, see
supplementary material,Supplementary Material online.
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NK Cluster Gene Organization the Hox cluster in the recently sequenced genome of the
In addition to Hox and ParaHox clusters, there is a further crustaceanDaphniapulex.Inthesecases,however,various
ANTP-classgeneclusterinsomeanimals,theNKclusterof losses or duplications of ParaHox genes in both O. dioica
arthropods.In Drosophila,theNKclustercontainstinman/ andarthropods(Daphniaandinsects),aswellasscattering
NK4,bagpipe/NK3,ladybird-earlyandladybird-late(together oftheHoxclusterinO.dioicathroughthesmallcompacted
being the fly representatives of Lbx), C15/Tlx, and slouch/ genome or the extensive genome rearrangements across
NK1(Jaglaetal.2001).Ancestrally,theinsectNKclusteralso thesequencedEcdysozoa(seeIntroduction),preventcon-
contained Msx/Drop and Hmx/NK5 (Garcia-Fernandez fident resolution of ancestral organization, as opposed to
2005; Richards et al. 2008). NK gene clustering in insects lineage-specific secondary associations. Consequently, we
isinmarkedcontrasttothesituationinchordates,inwhich favor the scenario in which the Hox and ParaHox clusters
the ancestral NK cluster has been broken up. Despite this were arranged on two separate chromosomes in the
disruption,inamphioxus(aproxyfortheancestralchordate PDA, an arrangement that has been conserved in both
D
condition),NK4,NK3,NK1,NK6,NK7,Lbx,Tlx,andMsxare Platynereis and amphioxus (fig. 3). ow
n
allon onechromosome (Lukeetal.2003;Butts et al. 2008; lo
a
Putnam etal. 2008; Wotton etal. 2009). The NK clusterof NK Genes: Comparable Breaks in d
e
d
the PDA thus probably consisted of nine NK genes (Msx, a Lophotrochozoan and Chordates and Ancient fro
NK4, NK3, Lbx, Tlx, NK7, NK6, NK1, and Hmx/NK5), with Separation of NK2 m
afivetosixgenecoretendingtoremainclusteredininsects ThePlatynereisNKclustergeneshavebeenseparatedinto http
(Buttsetal.2008).WemappedsevenNKclustergenesinP. s
dumerilii (PduMsx, PduNK4, PduNK3, PduLbx, PduTlx, tthrirgeuein‘sgelyg,mthenetps’oasliotinognsaosifntghleescehbroremaokssoimneth(efigP.la2tf–ynl)e.rIenis- ://aca
PduNK6, and PduNK1) and provide the first evidence that d
cluster are the same as those found in amphioxus, except e
m
theNKclustergenesarelocatedwithinasinglechromosome for a further separation of NK6 away from Lbx and Tlx in ic
in a lophotrochozoan (fig. 2f–l). In P. dumerilii, PduNK4, the Platynereis lineage (fig. 3). Perhaps shared regulatory .ou
p
PduNK3, and PduMsx are tightly linked together close to elementsorlong-rangeregulatorymechanismsactingover .co
the telomere-end of the long arm of the NK chromosome m
thesedistinctsubsetsoftheNKclustergenesmightbethe /m
(fig.2iandj).PduLbxandPduTlxarealsoclosetoeachother basis by which these subsets have been conserved in the be
butarelocatedclosetothecentromereofthelongarmof /a
theNKchromosome(fig.2fandg).PduNK1isthenfurther chordate and lophotrochozoan lineages. rtic
NK2genesarenotlinkedtoNKclustergenesinanyan- le
separated from these other NK cluster components and is -a
imalexaminedtodate,exceptforthemosquitoscrogene. b
located at the opposite telomere from PduMsx (fig. 2l). However, since A. gambiae has only three chromosomes, stra
c
the linkage of mosquito scro with the NK cluster genes t/2
Separation of the NK2 Genes 9
is most likely a secondary rather than ancient association. /1
NK2genesareanotherNKgenefamilyinanimals,butthese /1
Therefore, we suggest that separation of the NK2 family 5
7
havenotbeenaccommodatedintheMega-clusterhypoth- from the ancestral ProtoNK cluster-bearing chromosome /1
7
esis until now. In P. dumerilii, the NK2 family (PduNK2.1, 4
pre-datesthePDA,eitherviaararetrans-duplicationsuch 8
PduNK2.2a, and PduNK2.2b) is on a distinct chromosome that the NK2 family was never in the Mega-cluster or the 459
from those bearing the NK cluster genes, the Hox-linked b
NK2familyoriginatedintheclusterandthentranslocated y
genes, and the ParaHox genes (fig. 2m, n, p, q and u). In g
away before the origin of the PDA (fig. 3). Chromosome- u
e
allanimalsexaminedtodate,NK2genesareonadifferent s
chromosome from the NK cluster genes, except for the sscoalvlee mthaepsepitnwgodpaotassifbroilmitiens.onbilaterian lineages may re- t on 2
scarecrow(scro) gene of the mosquito Anopheles gambiae, 1
N
whichweproposeisasecondaryassociationinthisinsect o
Genome Architecture and ANTP-Class v
(see Discussion). em
Macrosynteny
b
e
Discussion LinkageoftheANTP-classhomeoboxgenesonaselectfew r 2
0
chromosomesislikelytobeduetoacombinationofevo- 1
8
Hox and ParaHox Clusters Were not Linked in the lutionary history (e.g., descended from tandem arrays)
PDA combined with functional constraints, such as those
The location of Hox and ParaHox cluster loci on distinct known for the long-range regulation of subsets of these
chromosomesinPlatynereisandamphioxus,andbyexten- genes such as the Hox cluster. Additionally, these genes
sionthePDA,impliesthatiftheParaHoxclusteroriginated may well be targets of common regulatory factors which
via a large tandem duplication, then this must have been may have led to their retention in particular regions of
followed by translocation before divergence of the proto- thegenome(Jangaetal.2008)oraresubjecttosomeother
stome and deuterostome lineages. In contrast to the form of nonrandom distribution constraint that is poorly
Platynereis/amphioxus situation, some instances of Hox understoodatpresent(Hurstetal.2004;BatadaandHurst
andParaHoxgenelinkagesareseeninotherinvertebrates, 2007; Koonin 2009). This in turn may account for the un-
suchaslinkageofOdiCdxtoOdiHox1(Seoetal.2004)inthe usualoccurrenceoftheposteriorHoxgene(PduPost1)re-
urochordate Oikopleura dioica, and the linkage of Cdx to locating to the NK chromosome in P. dumerilii. The
162
MBE
Annelid to Chordate Homeobox Macrosynteny · doi:10.1093/molbev/msr175
D
o
w
n
lo
a
d
e
d
fro
m
aFmIG.p3h.ioSxuumsmBraarnycohfioAstNomTPa-cfllaosrsidhaoe.mCeoolboorxcoodrginagniazastiinonfigounrteo1o.nDlyiafgoounralchlirnoemsoinsdoimcaetseinlartgheedinisftearnrceedsPbDeAtw,eaennnegleidnePslabtuyntewreitishdcuhmroemriloiis,oamnde https
linkagemaintained,indicatedbyhorizontallines.TheHoxclusterisdenotedasanelongatedboxtorepresentthepresenceofmultiplegenes, ://a
c
withvarying genenumber betweenthe threerepresentations. a
d
e
m
ic
translocation of this gene may have been constrained by NK clusters in a Mega-cluster. If this were the case, how- .o
u
p
aneedtoexistinagenomicregionconducivetoitsappro- ever, it is odd that none of the other Hox-linked or NK .c
o
priate and viable regulation. Other possibilities for this cluster and NK-linked genes are on this Daphnia scaffold, m
/m
arrangement include inheritance from a Mega-cluster ar- includingEvx,Dlx,Mox,En,Mnx,Gbx,NK1,orNK5,atleast
b
e
rangement with the Hox and NK clusters linked (unlikely, some of which would be expected in the intervening dis- /a
asanotherlophotrochozoanPost1,inLottiagigantea,isstill tance.Also,theextensivemacrosyntenythatweshowbe- rtic
le
withintheHoxcluster)orachancerelocationofPduPost1 tweenPlatynereisandchordatesimpliesthatthisDaphnia -a
b
to the NK cluster chromosome. organization is secondarily derived from a highly rear- s
tra
ThepossibilitythattranslocatingANTP-classhomeobox rangedancestorsomewhereinecdysozoanevolution.This c
genesmaymovetoanonrandomlocationhasmajorimpli- wouldalsobeconsistentwiththeunusualorganizationof t/29
/1
cationsfortheMega-clusterhypothesis.Thishypothesisis the Daphnia genome, with one chromosome constituting /1
5
based on a supposed NK-like gene (Dlx) being linked to 25%ofthetotalnuclearDNAandbeingmuchlongerthan 7
/1
theHoxcluster.WhetherornotDlxreallyisanNK-likegene alloftheremainingchromosomes(Colbourneetal.2011). 7
4
8
or not is one problem for the Mega-cluster hypothesis Of the remaining chromosomes, another 30% of the nu- 4
5
(see above). A second problem is that if Dlx did begin life clearDNAisfoundonchromosomes2–4,sothatonlyfour 9 b
y
withtheNKclusterandnotwiththeHoxcluster,thenthere of the 12 Daphnia chromosomes contain approximately g
u
may be a very limited range of genomic locations that it 55%ofthenuclearDNA.Thisimpliesthatthereareanum- e
s
could translocate to. Thus, Dlx linkage to the Hox cluster berofgenelinkagesthatresultfromsecondaryassociations t o
n
may not be indicative of (or the result of descent from) evolvingontheDaphnialineage,whichwerenotpresentin 2
1
a linkage of Hox- and NK-related genes but instead may an ancestor with genes distributed more uniformly across N
o
reflect Dlx translocating from the NK array and being the karyotype. A confident understanding of the linkages ve
m
restrictedtoasmallnumberoflocationsinwhichitcould seeninD.pulexrequirestheavailabilityofmoreecdysozo- b
e
be viably accommodated, one of which is the Hox angenomes,particularlythosewithhaploidnumbersthat r 2
0
chromosome. have not been greatly reduced by chromosome fusions. 1
8
Further evidence may come from diploblast genomes.
The Status of the Mega-Cluster Hypothesis Diploblastandchordategenomesshowsurprisinglyhighlev-
Given the above possibility of constrained genome archi- els of synteny (Putnam et al. 2007; Srivastava et al. 2008),
tecture restricting the viable destinations of translocating whichcontrastswithanalmostcompletelackofsyntenybe-
genes, is there then any evidence that a Mega-homeobox tween insects, nematodes, and chordates (Zdobnov et al.
cluster existed at all? The recently sequenced genome of 2005).Comparisonsperformedbetweengenomesofthecni-
anoninsectarthropod,Daphniapulex(whichhasahaploid darian Nematostella vectensis and vertebrates allowed the
numberof12),isintriguing,asaproportionoftheNKclus- identification of Putative Ancestral Linkage groups (PALs)
tergenes(NK4,NK3,Lbx,andTlx)areonthesamescaffold of theancestraleumetazoan (Putnametal.2007).Unfortu-
as the Hox cluster of D. pulex, as is the only remaining nately, these PALs do not accommodate many of the
Daphnia ParaHox gene, Cdx. Perhaps, this linkage is evi- Nematostella ANTP-class gene-containing scaffolds and do
dence for the ancient linkage of the Hox, ParaHox, and not reveal any Hox-linked and NK-linked associations that
163
MBE
Hui et al. · doi:10.1093/molbev/msr175
unambiguously resolve the veracity of the Mega-cluster hy- the 550-million-year-old PDA arrangement of the ANTP-
pothesis, except for one possible exception. Nematostella class homeobox genes is that they were split into four
scaffold 3 is part of the Hox PAL, and as well as containing distinct chromosomes: the Hox, ParaHox, NK, and NK2,
Hoxgenes,alsocontainsMsx,acomponentoftheancestral an arrangement that persisted to the present day in the
bilaterianNKcluster(seeabove).Thisthenmaybearemnant Platynereis and amphioxus lineages.
from, and evidence for, an ancestral Mega-cluster. Chromo-
some-levelmappinginNematostellaandfurtherdiploblastge- Supplementary Material
nomesassembledtolargecontigsizeswillbeneededtotest
Supplementary text, figures S1 and S2, and table S1 are
thisspeculation.NotwithstandingthelimitsoftheNematos- available at Molecular Biology and Evolution online
tella data in the present context,Putnam et al. (2007) spec- (http://www.mbe.oxfordjournals.org/).
ulatedthatonlyafewbreaksorfusionsperchromosomehad
happenedsincetheeumetazoanancestorintheNematostella Acknowledgments
D
andchordatelineages.OurPlatynereisdatareinforceandex- ow
Workintheauthors’laboratoriesissupportedbytheRoyal n
tendthisview.Platynereisalsohasaveryconservativegenome lo
Society and the Biotechnology and Biological Sciences Re- a
and the ecdysozoan models of arthropods and nematodes d
search Council . J.H.L.H. was supported by the Hong Kong ed
have undergone extensive genome rearrangements that are andChinaOxford ScholarshipFundsandtheMertonCol- fro
not typical for protostomes as a whole. m
legeDomusscholarshipandC.M.wassupportedbyaCom- h
Thegenome-levelorganizationoftheANTP-classhomeo- monwealth scholarship. Author Contributions: J.H.L.H., ttp
boxgenesisthusmostlikelytohaveevolvedviaapre-PDA C.M.,andD.E.K.F.wrotethemanuscript.D.E.K.F.conceived, s://a
Mega-cluster stage, or, a state close to a Mega-cluster but c
designed,andsupervisedtheproject.J.H.L.H.preparedchro- a
withonlyveryfewbreaksbeforetheevolutionofallthecon- de
mosomematerial,performedRACEandFISH,andisolated m
stituent homeobox gene families (the weak Mega-cluster most of the BAC clones; C.M. isolated the Dlx BAC clones ic.o
scenario, see Introduction). If this weak Mega-cluster u
and performed the Dlx FISH; A.S.M. isolated Hox BAC p
scenario is true, then there were only a maximum of three .c
clones;D.A.andG.B.providedcDNAclones;G.B.provided o
m
breaks(eithernontandemduplicationsorpre-Mega-cluster animals; P.W.H.H. co-supervised the study. All authors ap- /m
translocations) prior to the PDA, to produce the four b
proved the final version of the manuscript. e
saenpdarNatKe2csotmillpsoeneennitns;PthlaetyPnaerraeHisoaxn,dHoaxm-lpinhkieodx,uNs.KW-lihnaktedis, References /article
clear is that there is a surprisingly high degree of similarity -ab
oftheorganizationofANTP-classgenesbetweenPlatynereis Arendt D, Tessmar-Raible K, Snyman H, Dorresteijn AW, stra
WittbrodtJ.2004.Ciliaryphotoreceptorswithavertebrate-type c
and the chordate ancestor, which is inconsistent with opsinin aninvertebrate brain.Science 306:869–871. t/2
a secondary coming together or clustering of these genes BatadaNN,HurstLD.2007.Evolutionofchromosomeorganization 9/1
from widely scattered locations in an ancestral genome. driven by selection for reduced gene expression noise. Nat /15
7
Genet.39:945–949. /1
7
Conclusions Brooke NM, Garcia-Ferna`ndez J, Holland PWH. 1998. The ParaHox 4
8
gene cluster is an evolutionary sister of the Hox gene cluster. 45
Fossil data show that the major diversification of animal 9
Nature392:920–922. b
phyla, including polychaetes, occurred by the end of the Butts T, Holland PWH, Ferrier DEK. 2008. The urbilaterian Super- y g
Cambrianperiod(ConwayMorrisandPeel2008).Thepoly- Hoxcluster. TrendsGenet.24:259–262. ue
s
chaete P. dumerilii is hypothesized to have retained many Castro LF, Holland PWH. 2003. Chromosomal mapping of ANTP t o
primitivemorphologicalcharacteristicsofthePDA,includ- classhomeoboxgenesinamphioxus:piecingtogetherancestral n 2
ing ancestral cell types and developmental mode (Arendt genomes. EvolDev.5:459–465. 1 N
Castro LF, Rasmussen SL, Holland PWH, Holland ND, Holland LZ. o
et al. 2004; Tessmar-Raible et al. 2007; Dray et al. 2010). v
2006. A Gbx homeobox gene in amphioxus: insights into e
m
Atthegenomiclevel,thisanimalhasalsoproventobeless ancestry of the ANTP class and evolution of the midbrain/ b
e
derived than several other invertebrate systems, retaining hindbrainboundary. DevBiol. 295:40–51. r 2
0
a high proportion of ancestral introns (Raible et al. 2005) ColbourneJK,PfrenderME,GilbertD,etal.(69co-authors).2011.The 1
8
and elements of microsynteny (Hui et al. 2009). We now ecoresponsivegenomeofDaphniapulex.Science331:555–561.
Conway Morris S, Peel JS. 2008. The earliest annelids: Lower
reveal a strikingly low level of homeobox gene rearrange-
Cambrian polychaetes from the Sirius Passet Lagersta¨tte, Peary
ment and conserved macrosynteny with chordates. These
Land, NorthGreenland. ActaPalaeontol Pol.53:137–148.
dataimplythattheP.dumeriliilineageevolvedsignificantly
Coulier F, Popovici C, Villet R, Birnbaum D. 2000. MetaHox gene
slower than many other protostome lineages since the clusters. JExp Zool.288:345–351.
Cambrian. Although it remains a distinct possibility that DrayN,Tessmar-RaibleK,LeGouarM,etal.(10co-authors).2010.
theancestralMega-clusterexistedandthePDAcontained Hedgehogsignalingregulatessegmentformationintheannelid
thevastmajorityofthehypotheticalMega-clustercompo- Platynereis. Science 329:339–342.
Ferrier DEK. 2008. When is a Hox gene not a Hox gene? The
nentsonasinglechromosome,weerronthesideofcaution
importance of gene nomenclature. In: Minelli A, Fusco G,
here until more genome sequences are available along
editors. Evolving pathways: key themes in evolutionary de-
with further chromosome-level mapping work similar to velopmental biology. Cambridge (MA): Cambridge University
that described here. Our conservative interpretation of Press.p175–193.
164
MBE
Annelid to Chordate Homeobox Macrosynteny · doi:10.1093/molbev/msr175
FonsecaNA,VieiraCP,HollandPWH,VieiraJ.2008.Proteinevolution Putnam NH, Butts T, Ferrier DEK, et al. (37 co-authors). 2008. The
ofANTPandPRDhomeoboxgenes.BMCEvolBiol.8:200. amphioxus genome and the evolution of the chordate
Garcia-Ferna`ndez J. 2005. The genesis and evolution of homeobox karyotype. Nature 453:1064–1071.
gene clusters. NatRevGenet. 6:881–892. Putnam NH, Srivastava M, Hellsten U, et al. (19 co-authors).
Holland LZ, Albalat R, Azumi K, et al. (64 co-authors). 2008. The 2007. Sea anemone genome reveals ancestral eumetazoan
amphioxus genome illuminates vertebrate origins and cepha- gene repertoire and genomic organization. Science
lochordate biology. Genome Res.18:1100–1111. 317:86–94.
Howard-Ashby M, Materna SC, Brown CT, Chen L, Cameron RA, RaibleF,Tessmar-RaibleK,Osoegawa,etal.(12co-authors).2005.
Davidson EH. 2006. Identification and characterization of homeo- Vertebrate-type intron-rich genes in the marine annelid
boxtranscriptionfactorgenesinStrongylocentrotuspurpuratus,and Platynereis dumerilii. Science 310:1325–1326.
theirexpressioninembryonicdevelopment.DevBiol.300:74–89. RichardsS,GibbsRA,WeinstockGM,etal.(191co-authors).2008.
HuiJH,RaibleF,KorchaginaN,etal.(11co-authors).2009.Features ThegenomeofthemodelbeetleandpestTriboliumcastaneum.
of the ancestral bilaterian inferred from Platynereis dumerilii Nature 452:949–955.
ParaHox genes. BMC Biol.7:43. RyanJF,BurtonPM,MazzaME,KwongGK,MullikinJC,FinnertyJR. D
o
Hurst LD, Pal C, Lercher MJ. 2004. The evolutionary dynamics of 2006. The cnidarian-bilaterian ancestor possessed at least 56 w
n
eukaryotic geneorder. NatRev Genet.5:299–310. homeoboxes:evidencefromthestarletseaanemone,Nematos- lo
a
Jagla K, Bellard M, Frasch M. 2001. A cluster of Drosophila tella vectensis.Genome Biol.7:R64. d
e
homeobox genes involved in mesoderm differentiation pro- Ryan JF, Mazza ME, Pang K, Matus DQ, Baxevanis AD, d
grams. Bioessays 23:125–133. Martindale MQ, Finnerty JR. 2007. Pre-bilaterian origins of the fro
m
Janga SC, Collado-Vides J, Babu MM. 2008. Transcriptional HoxclusterandtheHoxcode:evidencefromtheseaanemone, h
regulation constrains the organization of genes on eukaryotic Nematostella vectensis. PLoSOne 2:e153. ttp
s
chromosomes. ProcNatl AcadSci USA. 105:15761–15766. Seo HC, Edvardsen RB, Maeland AD, et al. (12 co-authors). 2004. ://a
JhaAN,HutchinsonTH,MackayJM,ElliotBM,PascoePL,DixonDR. Hoxclusterdisintegrationwithpersistentanteroposteriororder c
a
1995. The chromosomes of Platynereis dumerilii (Polychaeta: ofexpression inOikopleura dioica. Nature 431:67–71. de
m
Nereidae). J MarBiolAssocUK.75:551–562. SrivastavaM,BegovicE,ChapmanJ,etal.(21co-authors).2008.The
ic
Koonin EV. 2009. Evolution of genome architecture. Int J Biochem Trichoplax genome and the nature of placozoans. Nature .o
u
Cell Biol.41:298–306. 454:955–960. p
.c
Larroux C, Fahey B, Degnan SM, Adamski M, Rokhsar DS, and Takatori N, Butts T, Candiani S, Pestarino M, Ferrier DEK, Saiga H, o
m
DegnanBM.2007.TheNKhomeoboxgeneclusterpredatesthe HollandPWH.2008.Comprehensivesurveyandclassificationof /m
origin ofHoxgenes. Curr Biol.17:706–710. homeobox genes in the genome of amphioxus, Branchiostoma be
Luke GN, Castro LF, McLay K, Bird C, Coulson A, Holland PWH. floridae.DevGenes Evol. 218:579–590. /a
2003. Dispersal of NK homeobox gene clusters in amphioxus Tessmar-Raible K, Raible F, Christodoulou F, Guy K, Rembold M, rtic
le
and humans.Proc NatlAcad SciU SA.100:5292–5295. Hausen H, Arendt D. 2007. Conserved sensory-neurosecretory -a
b
MillerDJ,MilesA.1993.Homeoboxgenesandthezootype.Nature cell types in annelid and fish forebrain: insights into s
365:215–216. hypothalamus evolution. Cell 129:1389–1400. tra
c
Minguillo´nC,Garcia-Ferna`ndezJ.2003.Genesisandevolutionofthe Wotton KR, Weierud FK, Juarez-Morales JL, Alvares LE, Dietrich S, t/2
9
Evx and Mox genes and the extended Hox and ParaHox gene Lewis KE. 2009. Conservation of gene linkage in dispersed /1
clusters. Genome Biol.4:R12. vertebrate NK homeoboxclusters 219:481–496. /15
7
Monteiro AS, Schierwater B, Dellaporta SL, Holland PWH. 2006. A Zdobnov EM, Bork P. 2007. Quantification of insect genome /1
low diversity of ANTP class homeobox genes in Placozoa. Evol divergence. TrendsGenet. 23:16–20. 74
8
Dev.8:174–182. ZdobnovEM,vonMeringC,LetunicI,BorkP.2005.Consistencyof 4
5
Pollard SL, Holland PWH. 2000. Evidence for 14 homeobox genome-basedmethodsinmeasuringMetazoanevolution.FEBS 9 b
geneclustersinhumangenomeancestry.CurrBiol.10:1059–1062. Lett. 579:3355–3361. y
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