Article

The Loss of Adipokine Genes in the Chicken Genome andImplications for Insulin MetabolismNatasa -Dakovicy1234 Morgane Terezoly1234 Frederique Pitel5 Virginie Maillard1234 Sebastien Elis1234

Sophie Leroux5 Sandrine Lagarrigue6 Florence Gondret6 Christophe Klopp7 Elisabeth Baeza8

Michel J Duclos8 Hugues Roest Crollius91011 and Philippe Monget1234

1UMR85 Physiologie de la Reproduction et des Comportements INRA Nouzilly France2UMR7247 CNRS Nouzilly France3Universite Francois Rabelais de Tours Tours France4Institut Francais du Cheval et de lrsquoEquitation Nouzilly France5UMR1388 INRAINPT ENSATINPT ENVT Genetique Physiologie et Systemes drsquoelevage INRA Castanet Tolosan France6INRA Agrocampus - Ouest UMR1348 Physiologie Environnement et Genetique pour lrsquoAnimal et les Systemes drsquoelevageSaint-Gilles7INRA SIGENAE Toulouse France8INRA UR83 Recherches Avicoles Nouzilly France9Ecole Normale Superieure Institut de Biologie de lrsquoENS IBENS Paris France10CNRS UMR 8197 Paris France11Inserm U1024 Paris FranceyThese authors contributed equally to this work

Corresponding author E-mail PhilippeMongettoursinrafr

Associate editor Shizhong Xu

Abstract

Gene loss is one of the main drivers in the evolution of genomes and species The demonstration that a gene has been lostby pseudogenization is truly complete when one finds the pseudogene in the orthologous genomic region with respect toactive genes in other species In some cases the identification of such orthologous loci is not possible because ofchromosomal rearrangements or if the gene of interest has not yet been sequenced This question is particularlyimportant in the case of birds because the genomes of avian species possess only about 15000 predicted genes incomparison with 20000 in mammals Yet gene loss raises the question of which functions are affected by the changes ingene counts We describe a systematic approach that makes it possible to demonstrate gene loss in the chicken genomeeven if a pseudogene has not been found By using phylogenetic and synteny analysis in vertebrates genome-widecomparisons between the chicken genome and expressed sequence tags RNAseq data analysis statistical analysis ofthe chicken genome and radiation hybrid mapping we show that resistin TNF and PAI-1 (SERPINE1) three genesencoding adipokines inhibiting insulin sensitivity have been lost in chicken and zebra finch genomes Moreover omentina gene encoding an adipokine that enhances insulin sensitivity has also been lost in the chicken genome Overall only oneadipokine inhibiting insulin sensitivity and five adipokines enhancing insulin sensitivity are still present in the chickengenome These genetic differences between mammals and chicken given the functions of the genes in mammals wouldhave dramatic consequences on chicken endocrinology leading to novel equilibriums especially in the regulation ofenergy metabolism insulin sensitivity as well as appetite and reproduction

Key words chicken adipokines insulin resistance

IntroductionNotable differences exist in the regulation of energy metabo-lism between chicken and mammals In mammals and inparticular human increased body mass index is frequentlyassociated with a decrease in insulin sensitivity leading toinsulin and type II diabetes (Bray and Bellanger 2006 Hajeret al 2008) and altered plasma profiles at higher leptin andlower adiponectin levels (Finucane et al 2009 Barazzoni et al2012) Insulin circulates at comparable levels in chickens andmammals but chickens are less sensitive to insulin action

(Simon 1989) and glucose circulates in birds at muchhigher level than in mammals (2 gl vs 1 gl respectively)This refractoriness is particularly evident in adipose tissues(Dupont et al 2012) In an experimental selection for fatnessin chickens no insulin resistance was observed but only animbalance in glucose versus insulin with lower glucose andhigher insulin levels (Touchburn et al 1981 Simon andLeclercq 1982) and an increased activation of the insulin cas-cade in the liver (the major site of lipogenesis in chicken) of fatchickens (Dupont et al 1999) Although insulin resistance can

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be induced by corticosterone treatment of chickens (Taouiset al 1993 1996) to our knowledge there is no report ofspontaneous insulin resistance or type II diabetes in this spe-cies In mammals insulin sensitivity is notably regulated bythe action of adipokines hormones secreted by adiposetissue which are able to modulate the activity of the insulinreceptor (Steppan et al 2005 Mao et al 2006 Wang et al2009 Jacques et al 2012) Among the subset of analyzedadipokines seven are known to enhance insulin sensitivity(leptin omentin visfatin adiponectin vaspin chemerinand apelin) whereas four others decrease insulin sensitivity(IL6 TNF PAI-1 [SERPINE1] and resistin) (Fantuzzi 2005Alessi and Juhan-Vague 2006 Chang et al 2011 Kwon andPessin 2013)

Here we analyze the chicken genome in an attempt toprove the presence or absence of some of the genes impor-tant for the above mentioned functions It is however diffi-cult to demonstrate that a gene has been lost from a genomeIdeally the presence of an inactivated copy of a gene which isunable to lead to a functional protein because of the presenceof premature stop codons or loss of open reading frame andwhich is located in the orthologous genomic region with re-spect to active genes in other species is considered sufficientevidence of gene loss However in some cases the identifica-tion of such orthologous loci is difficult because chromosomalrearrangements tend to reorganize genomes and erase thesignals of conservation in the locus of interest particularlybetween species separated by long evolutionary distancesAlternatively the gene of interest may be located in a geno-mic region that is not yet sequenced Sequence coverage forthe autosome chromosomes in chicken was estimated to be98 and gene coverage 90ndash95 (Burt 2006) About 5ndash10 ofthe protein-coding genes are still missing from the Ensemblchicken gene set with a very poor coverage of microchromo-somes particularly GGA16 and GGAW (Hiller et al 2004Griffin and Burt 2014) The genomes of avian species possessonly about 15000 predicted genes whereas in mammals agenome typically contains more than 20000 genes (Hughesand Friedman 2008) If this reflects the true state of thechicken genome and is not due to sequencing artifacts (be-cause several microchromosomes have not yet been se-quenced in birds) this raises the question of whichfunctions were affected by the changes in gene countseither through gene family amplification in mammals orgene losses in birds

The difficulty in concluding about the gene loss is illus-trated by the leptin gene Since the first publication on thecloning of a chicken leptin cDNA (Taouis et al 1998) severalstudies have described the in vitro or in vivo regulation of itsexpression as well as its biological roles in this species (Taouiset al 2001 Dridi et al 2005) However these results led to acontroversy that questioned whether the gene existed in thechicken genome at all (Sharp et al 2008 Simon et al 2009)We have previously brought forward several strong argu-ments for the loss of the leptin gene in the chickengenome resulting from the deletion of a 1-Mb genomicregion (Pitel et al 2010) without detectable presence of apseudogene

By using a phylogenetic and synteny analyses genome-wide comparisons between the chicken genome and ex-pressed sequence tag (ESTs) RNAseq data analysis statisticalanalysis of the chicken genome and radiation hybrid (RH)mapping we show in this study that three genes encodingadipokines inhibiting insulin sensitivity (resistin TNF andPAI-1) and one gene encoding an adipokine that enhancesinsulin sensitivity (omentin) have been lost in the chicken andzebra finch genomes

Results

Identification of Adipokine-Encoding Genes PutativelyLost in Bird Genomes

We chose to study the evolution of 11 genes known toencode adipokines in mammals Leptin IL6 resistin TNFPAI-1 adiponectin omentin apelin visfatin vaspin and che-merin By systematically analyzing phylogenetic trees of thesegenes in the Ensembl database we found that five were pre-sent in fish and mammalian genomes but absent in birdsLeptin as shown previously (Pitel et al 2010) resistin TNFPAI-1 and omentin (fig 1) In addition leptin and resistin arealso lost in all sauropsids We thus systematically looked fortheir pseudogenes in syntenic regions and did not find any

Characterization of Deleted Genomic Regions inChickenmdashLoss of Resistin TNF PAI-1 and OmentinGenes

We only describe here the approach for one locus centeredon the resistin gene because we have applied the same pro-cedure for the four loci (and similarly to the previous analysisof the leptin gene (Pitel et al 2010) The human resistin geneis localized on chromosome 19 in a synteny block well con-served within mammals Genes that are part of this humanblock (from 7585 to 7987 Mb) that contains the resistin geneis not annotated in the current chicken sequence assemblyMore precisely this block contains 26 genes of which 15 do

FIG 1 The birth and loss of four adipokine genes studied here as well asleptin (previously described by Pitel et al 2010) Different genes arerepresented by different symbols in different colors The birth of agene is depicted by a solid symbol and the loss of a gene is depictedby a hollow symbol

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not match by reciprocal tBLASTn analysis on the chickengenome nor on chicken ESTs (supplementary table S1Supplementary Material online) The four genes MCOLN1PNPLA6 TRAPPC5 and EVI5L match with ESTs by reciprocaltBLASTn analysis and are annotated in the chicken genomebut in unplaced contigs whereas XAB2 matched only chickenESTs To confirm that these five genes are not located nearthe chicken orthologs of gene at boundaries of this blockin the human genome (ARHGEF18 PEX11G and C19Orf45on the telomeric side and CTXN1 TIMM44 and ELAVL1 onthe centromeric side) experiments on RH panels were per-formed (fig 2) On one side of the locus ARHGEF18 andC19Orf45 are confirmed to be linked to markers on chickenchromosome 28 CTXN1 and ELAVL1 map near MNT-346also a GGA28 (Gallus gallus chromosome 28) marker onthe other side of the locus Between these two blocks weused the available sequences to design primer pairs amplifyingfive other genes (MCOLN1 PNPLA6 XAB2 TRAPPC5 andEVI5L) All the genes map to chicken chromosome 30 (seeChickRH web server chickrhtoulouseinrafr) in two groupsMCOLN1 and PNPLA6 and XAB2 TRAPPC5 and EVI5L (fig 2)We thus show here that orthologs of the genes from the

resistin locus on HSA chromosome 19 are not in a singleconserved locus in the chicken genome because the bound-aries of the blocks are on chicken chromosome 28 whereasthe rest of the block is separated into at least three indepen-dent blocks in the chicken genome on other chromosomes

As shown in supplementary table S1 SupplementaryMaterial online similar genomic rearrangements of thisregion also seem to have occurred in the zebra finchgenome except for EVI5L residing on zebra finch chromo-some 8 Interestingly in lizard the missing genes are lessnumerous

As for the resistin example we performed the same RHmapping experiments for markers located near the threeother studied genes (TNF PAI-1 and omentin) in thehuman genome In each case the result confirmed theabsence of synteny between the chicken and the humangenomes in the region of interest (figs 3ndash5)

Overall our results show that the chicken genome havelost 5 out of 11 adipokines omentin TNF PAI-1 resistin(supplementary table S1 Supplementary Material online)and leptin (Pitel et al 2010) Interestingly the genes encodingthe receptors of leptin (leptin receptor LEPR NP_9896541)TNF (tumor necrosis factor receptor type 1-associatedDEATH domain protein TRADD XP_4140671) and resistin

FIG 2 Comparative map of the resistin genomic region Conservedsynteny between human chromosome 19 (HSA19) and the chicken(GGA) genome obtained through RH mapping The map is given inMb from assembly version GRCh37hg19 The color corresponds to thegene fragments in supplementary table S1 Supplementary Materialonline

FIG 3 Comparative map of the omentin genomic region Conservedsynteny between human chromosome 1 (HSA01) and the chicken(GGA) genome obtained through RH mapping The map is given inMb from assembly version GRCh37hg19 The color corresponds to thegene fragments in supplementary table S1 Supplementary Materialonline un unlinked to USF1 or PVRL4

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(adenylate cyclase-associated protein 1 CAP1XP_0036426321) are present in the chicken genome

Characterization of Adipose Tissue RNA-SeqDatamdashNo Transcript for Resistin TNF PAI-1 andOmentin

We identified 14385 genes in abdominal chicken adiposetissue with at least ten reads on average per bird amongthem 12341 referenced genes and 2044 potentially newgenes not yet annotated in the Ensembl reference genomeAmong the referenced genes expressed we observed visfatinand adiponectin with 1204 and 4247 reads per bird respec-tively showing a relatively high level of expression in the ad-ipose tissue of the 14-week-old chickens In contrast none ofthe 2044 new genes expressed in the chicken adipose tissuematched by reciprocal tBLASTn analysis on human or mouseresistin TNF PAI-1 or omentin gene sequences further sup-porting the hypothesis that these genes are not present in thechicken genome The de novo assembly of the RNA-Seq readsproduced 57875 contigs (total length 85 084733 bp N50

3550 bp) The bidirectional comparison of these contigs withchicken RefSeq transcripts shows that no transcripts corre-sponding to resistin TNF PAI-1 and omentin were foundwith our RNA-Seq data after de novo assembly The RNA-Seqresults reinforce the conclusion that the genes encoding resis-tin TNF PAI-1 and omentin were lost the chicken genome

Chromosomal Rearrangements of the RegionContaining Resistin Gene in Mammals Comparedwith Sauropsids

We took advantage of the Genomicus genome browser(Muffato et al 2010) to provide a better understanding ofthe evolution of the organization of the genomic region thatcontains the resistin gene in mammals in comparison withchicken zebra finch and lizard Thus taking the humangenome as a reference we have focused on the 75ndash8 Mbregion depicted in supplementary table S1 SupplementaryMaterial online In supplementary figure S1 SupplementaryMaterial online we propose a model of chromosomal evolu-tion with this region being divided into seven fragments de-fined by their gene content and gene order The position ofthe corresponding genes in human dog chicken zebra finch

FIG 4 Comparative map of the TNF- genomic region Conservedsynteny between human chromosome 6 (HSA06) and the chickengenome obtained through RH mapping The map is given in Mbfrom assembly version GRCh37hg19 The color corresponds to thegene fragments in supplementary table S1 Supplementary Materialonline

FIG 5 Comparative map of the PAI-1 (SERPINE1) genomic regionConserved synteny between human chromosome 7 (HSA07) and thechicken (GGA) genome obtained through RH mapping The map isgiven in Mb from assembly version GRCh37hg19 The color corre-sponds to the gene fragments in supplementary table S1Supplementary Material online

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and lizard is presented in supplementary table S1Supplementary Material online The order of the seven frag-ments is well conserved from the ancestor of amniota tohuman and dog (mammals in general) and is subjected todramatic rearrangements in lizard with conserved fragments2 4 5 and 6 (with a paracentric inversion of fragment 4 andan insertion within the fragment 2) In contrast chicken andzebra finch only conserve fragments 1 and 7 (with paracentricinversion of fragment 1) one or two other fragments beinginserted in between

DiscussionIn this study we propose a methodological approach to dem-onstrate that a gene is lost by deletion of a genomic regionThis approach combines reciprocal tBLASTn analysis of mam-malian genes on the chicken genome and ESTs with RHmapping of genes which are detected by reciprocaltBLASTn on the genome but are not localized on a chromo-some and ESTs Among human genes present in the humangenomic region ldquosyntenic with deleted chicken genomiclocus some have their chicken ortholog localized on anotherchromosome (or have not yet been localized) suggesting thatseveral of these chicken genes have not been lost but ratherhave been ldquodispersedrdquo in chicken genome Concerning theresistin locus there is no synteny conservation betweenhuman chromosome 19 and the chicken genome at the chro-mosome level but there are several small synteny groupsmainly localized on chicken microchromosomes or unknownlinkage groups as previously observed (Morisson et al 2007)The omentin gene maps to human chromosome 1 in a regionwith a high number of intrachromosomal rearrangements onchicken chromosome 25 (Douaud et al 2008) Similarly theTNF gene maps to human chromosome 6 bearing themajor histocompatibility complex The homologous regionin chicken is localized on GGA16 a microchromosome withvery few sequence data when compared with other se-quenced microchromosomes due to compositional reasonsand already known showing many rearrangements comparedwith the human genome (Solinhac et al 2010)

Our results also suggest that most but not all of the genesstudied here that have been lost in the chicken genome havealso been lost in zebra finch This leads to the hypothesis thatthe loss of these genes may have occurred early in the evo-lution of birds However recent studies have reported pres-ence of a leptin gene in several wild birds including the zebrafinch (Friedman-Einat et al 2014 Huang et al 2014 Prokopet al 2014) Peregrine falcon genome sequence alignmentshowed high synteny alignment with human mouse andanother falcon species (Friedman-Einat et al 2014 Prokopet al 2014) Friedman-Einat et al (2014) also used thenewly identified LEP sequences in the dove Tibetan groundtit zebra finch and falcons as a query sequences for identifi-cation of LEP gene in the chicken turkey and duck genomesHowever no significant sequence similarity to LEP was foundwhich gives strong support that the LEP gene is missing fromthe chicken genome and in other domesticated poultry spe-cies The presence of LEP gene in wild birds (such as falconrock dove and zebra finch) (Friedman-Einat et al 2014

Prokop et al 2014 Huang et al 2014) but not in domesticatedbirds (Friedman-Einat et al 2014) suggests that LEP gene hasbeen lost in the process of domestication

Despite the high degree of interchromosomal conservationand conserved synteny between the human and chicken ge-nomes (Burt et al 1999 Groenen et al 2000) intrachromo-somal rearrangements are also common and associated withhigh recombination rates (Crooijmans et al 2001 Volker et al2010) The rearrangements that occurred in the lizardgenome were either quite similar with fewer deleted genesthan for birds (TNF and PAI-1) or markedly different(resistin) suggesting that there is a species-specific chromo-somal rearrangement and loss of genes during the evolutionof sauropsids The four loci studied are well conserved inmammals at least in human and dog Overall our resultsconfirm that the genome of sauropsids has undergone pro-found structural rearrangements after the divergence ofamniota species

In contrast the genes encoding the receptors of leptinTNF and resistin are present in the chicken genome increas-ing the list of orphan receptors This result constitutes a novelexample of a ldquobreakrdquo between ligands and receptors duringthe course of species evolution (Markov et al 2008) Theleptin and TNF receptors are functional because heterolo-gous ligands have biological effects on chicken in vitro andorin vivo (Adachi et al 2008 Takimoto et al 2008) In vivo onecan hypothesize that the loss of some of these adipokineswould be functionally compensated by paralogs This is pos-sible for adipokines that belong to families such as TNFwhich has two paralogs in the chicken genome FasL andTNFS15 Lipopolysaccharide-induced TNF known toinduce the expression of TNF in mammals is also able toinduce the expression of chicken TNFS15 in vitro (Hong et al2006) In the mouse targeted inactivation of TNF and its twomammalian close paralogs Lymphotoxin-a and -b (Lta andLtb absent in the chicken genome) showed that each paralogseems to play specific functions and has largely non redun-dant functions in vivo (Kuprash et al 2002) Functional com-pensation may also be possible for PAI-1 that has two paralogsin the chicken genome (SERPINE2 and SERPINE3) In particu-lar it has been shown that PAI-1-deficient mice and humanshave no spontaneous phenotype likely because other inhib-itors of the uPAplasmin system mask the effect of PAI-1defect In contrast to TNF and PAI-1 omentin and resistingenes have one paralog in mammals (omentin-2 and resistinlike respectively) but none in chickens So the consequence oftheir loss in the chicken genome remains to be elucidated

Compared with mammals chickens have much higherplasma glucose levels (2 gl) despite the presence of hyperac-tive insulin (Hazelwood et al 1968 Simon 1989) They arehowever less sensitive to the action of insulin relative tomammals Nevertheless spontaneous insulin resistance isnot observed in this species In mammals insulin resistanceand obesity are more apparent in adults However insulinresistance in adult sexually mature chickens is not wellknown Most of the studies of insulin sensitivity in chickenswere performed on individuals after hatching up to 17 weeksof age (Simon and Leclercq 1982 Tokushima et al 2003

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Dupont et al 2008 2009) However Chou and Scanes (1988)showed that young male chickens were more sensitive toinsulin than the adult male chickens of the same strains(White Leghorn) They also showed that there was no differ-ence between young male chicks of broiler (meat-type breed)and White Leghorn (egg-type breed) strains (Chou andScanes 1988)

Our results show that chicken genome have lost 5 out of11 adipokines evaluated here leptin (Pitel et al 2010) omen-tin TNF PAI-1 and resistin Overall out of 11 adipokinesstudied here only 1 adipokine inhibiting insulin action IL-6and 5 adipokines enhancing insulin sensitivity apelin visfatinvaspin chemerin and adiponectin are still present in thechicken genome This could lead to a profound change ofendocrinological equilibrium more permissive to insulinaction in birds in comparison with mammals Because thereceptors of these lost adipokines are present in the chickengenome it would be particularly interesting to treat in vivochicken selected for fatness with one or several of humanadipokines that reduce the sensitivity to insulin (TNF andresistin) and to look for a possible effect on insulin resistanceIt would also be interesting to inhibit one or several of thecorresponding proteins in humans which suffer from type IIdiabetes and to look for a possible increase in insulin sensi-tivity and a reversion of the disease

The loss of these adipokines may also have significant con-sequences on the regulation of reproduction In mammalsadipokines appear to regulate the functions of the reproduc-tive axis (Campos et al 2008 Hausman et al 2012 Landryet al 2013 Dupont et al 2014) In particular leptin modulatesmRNA expression of Kiss-1 (Tena-Sempere 2006 Luque et al2007 Ahn et al 2012) which in turn stimulate GnRH andgonadotropin secretion (Caraty et al 2012 Pinilla et al 2012)In contrast to mammals the female reproductive physiologyof birds is characterized by the preovulatory release of LHwhich relies on progesterone secreted by preovulatory folliclesfor the initiation of a GnRH peak prior to ovulation (Johnsonand Leone 1985 Etches and Petitte 1990) Birds are the onlyspecies that have lost Kysspeptins (Um et al 2010 Kanda andOka 2013) and other mechanism such as positive feedbackby progesterone and not estrogens to induce the preovula-tory surge of GnRH seems to be central for the regulation ofreproduction Moreover it is possible that birds maintain atighter regulation of GnRH without the control by Kisspeptinsor leptin due to environmental cues (eg photoperiod)

The regulation of appetite is also very different in chickenIn mammals leptin partly exerts its anorexigenic effect via theincrease and decrease of proopiomelanocortin and neuro-peptide Y release respectively and ghrelin stimulates appe-tite In chicken in contrast to mammals there are no leptinand Kiss system and ghrelin inhibits food intake (Kaiya et al2013) It remains unknown whether other neuropeptidesandor circuitry replace leptin and Kiss peptide in functionsin the hypothalamus

Because several studies have shown that leptin and TNFhave biological effects on chicken in vivo and in vitro somelaboratories andor companies have developed assays to mea-sure the concentration of these peptides as well as of

omentin Although several technical reports have pinpointedthe fact that some antibodies (commercially available or pro-duced by laboratories) are not well characterized (Saper 2005Pradidarcheep et al 2008) and recognize several moleculesnot related to the molecule of interest the targets of anti-bodies against the product of missing genes remain to beidentified

This work has implications for comparative physiology andendocrinology In birds the absence of several genes reportedto control the energetic metabolism of mammals could ex-plain some species specificities such as hyperglycemia andrelative insensitivity to insulin action This basic informationcould contribute to a better understanding of the physiologyof metabolic regulations across species and of physiopatho-logical conditions such as human type II diabetes

Materials and Methods

General Methodological Approach to Study GeneLoss

To look for a possible loss of genes resistin (RETN) PAI-1omentin (ITLN1) and TNF in the chicken genome we per-formed reciprocal tBLASTn analyses httpblastncbinlmnihgovBlastcgi last accessed July 23 2014) on the sequence ofthe chicken genome and on chicken ESTs with the humanprotein sequence as a query (fig 6) We also used the dogprotein sequence as a control We searched for possible pseu-dogenes and inferred the presence of a pseudogene only if amatch was found by tBLASTn analysis in the syntenic locus incomparison with the other species of interest with a stopcodon or an indel in the sequence identified by the similaritysearch (Meslin et al 2012)

We performed reciprocal tBLASTn analyses on the chickengenome using the following general parameters Expectthreshold 10 word size 3 scoring matrix BLOSUM62 gapcost existence 11 extension 1 compositional adjustmentsconditional compositional scorematrix adjustments andfilter low complexity regions To assess whether a given align-ment constitutes evidence for homology and is not due to achance we checked the statistics of sequence similarityscores The statistics for positive reciprocal best hits was asfollows Max scores ranged between 63 and 554 E valueranged between 0 and 9e-156 and identity ranged between47 and 95 We have also checked sequences of shortlength with high E value to search for possible pseudogenes(Meslin et al 2012) The number of G gallus ESTs screenedwas 600434 (dbEST release 130101) from all available tissues

First we ran reciprocal tBLASTn analyses on the chickengenome (fig 6 step 1) with two possible outcomes Match orno match In case we obtained a positive reciprocal matchthat is two genes (in our study a human and a chicken gene)each in different genome find each other as their best respec-tive hit the gene was localized on a known or unknownchromosome Furthermore if the outcome was ldquono matchrdquowe performed reciprocal tBLASTn analyses on chicken ESTs(fig 6 step 2) for genes that are annotated in human but notin the chicken genome (no match) If no positive reciprocaltBLASTn sequence (no match) was found in ESTs we tested

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the hypothesis that the gene of interest and possible itsneighboring genes were deleted from the genome For thispurpose we defined the corresponding human syntenicregion centered on the gene of interest and bordered bythe first neighboring genes for which chicken orthologsexist and were localized in a syntenic region of the chickengenome (fig 6 step 3) For each of these genes we system-atically performed reciprocal tBLASTn analysis against thechicken genome and chicken ESTs (fig 6 step 4) Most fre-quently both tBLASTn analyses were negative (no match)and the genes were predicted to be deleted with the blockFor some genes reciprocal positive matches were foundagainst ESTs the gene being either localized in the chickengenome (in a non syntenic region) or predicted to be on anldquounknown chromosomerdquo In this case experiments onchicken RH panels were conducted to verify if these geneswere localized within the syntenic region (fig 6 step 5)

Finally we analyzed an RNA-Seq data set generated fromadipose tissue and characterized by high sequencing coverage(300 millions of mapped reads from 8 birds see below) tomodel new genes absent from the Ensembl database to verifythat these genes are not expressed in this tissue

RH Panel Analysis

RH mapping was performed for markers located near thegenes of interest in the human genome Polymerase chainreaction (PCR) amplifications were carried out for eachmarker with specific primers (supplementary table S2Supplementary Material online) in 15ml reactions containing

25 ng DNA from the chickRH6 panel (Morisson et al 2002)04mM of each primer 025 units Taq polymerase (GoTaqPromega) 15 mM MgCl2 and 02 mM dNTP on a GeneAmpPCR System 9700 thermocycler (Applied Biosystems) Thefirst 5-min denaturation step was followed by 35 cycleseach consisting of denaturation at 94 C for 30 s annealingat Tm for 30 s and elongation at 72 C for 30 s PCR productswere analyzed on 2 agarose gels electrophoresed in 1 TBEbuffer and visualized by staining with ethidium bromideMapping of the markers on the RH panel was per-formed through the ChickRH server (httpchickrhtoulouseinrafr last accessed July 21 2014) Distances and two-point LODs were calculated through the Carthagene software(de Givry et al 2005) Maps were drawn with MapChart 20(Voorrips 2002)

RNA-Seq Analysis

RNA-Seq data sequencing and analysis were performed fromabdominal adipose tissue of 8 male and female chickens of 14weeks of age with 40 million of reads expected per bird de-scribed by Roux P-F Fresard L Leroux S Klopp C Martin PDesert C Fabre S Esquerre D Dehais C Djari A Zerjal TGourichon D Pitel F Lagarrigue S (unpublished data) Tocheck if the genes resistin PAI-1 omentin and TNF canbe found in the adipose chicken transcriptome all the result-ing bam files were merged to produce a unique referencealignment on which the discovery of new genes and tran-scripts was performed using CUFFLINKS 200 (Trapnell et al2010) The resulting GTF file was used to extract the fasta

FIG 6 Methodological approach to study gene loss

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sequence of the newly discovered transcripts to annotate andquantify them for each bird In addition an RNA-Seq subsetwas assembled de novo (26 828416 reads pairs from adiposetissue from one individual bird because of the high calcula-tion resources required) The assembly produced 57875 con-tigs (total length 85084733 bp N50 3550 bp) The newtranscripts discovered from the first approach and the contigsobtained by the de novo assembly were aligned on thehuman RefSeq set using BLASTx (E-valuelt 1e-3) ftpftpncbinlmnihgovrefseqH_sapiensmRNA_Prothumanrnafnagz 654 MB 060114 173600) and the RefSeq set wasaligned on the new transcripts discovered and contigs usingtBLASTn (E-valuelt 1e-3) For the five sought proteins theranks of the hits in both alignments were compared with findthe possible best reciprocal BLAST hits

Multispecies Comparative Map

Genome fragments were defined according to their genecontent and order Their localization in human dog chickenzebra finch and lizard was determined from the NationalCenter for Biotechnology Information genome browser(httpwwwncbinlmnihgov last accessed July 23 2014)using the most recent assemblies (Build 363 for Homo sapi-ens Build 31 for Canis familiaris Build 40 for G gallus Build21 for Taeniopygia guttata and Build 11 for Anolis carolinen-sis) Comparative mapping results based on gene contentwere also confirmed by comparative sequence analysis ofthe region of interest available from the Ensembl (version71ndash74) (httpwwwensemblorg last accessed July 23 2014)and Genomicus (httpwwwgenomicusbiologieensfrgeno-micus-7201cgi-binsearchpl last accessed July 23 2014)genome browsers

Supplementary MaterialSupplementary tables S1 and S2 and figure S1 are available atMolecular Biology and Evolution online (httpwwwmbeoxfordjournalsorg)

Acknowledgments

This work was supported by FATINTEGER Agence NationaleRecherche (ANR) grant no ANR-11-BSV7-004 The authorsthank Pascal Martin from INRA Tox-alim laboratory and thePlateforme GENOTOUL Castanet Tolosan for generatingcDNA library and RNA-Seq raw data These data werefunded by INRA in the scope of SOSRNASeq program (2010)

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ceptor is functional in activating JAK-STAT pathway in vitro JEndocrinol 197335ndash342

Ahn SY Yang SW Lee HJ Byun JS Om JY Shin CH 2012 Excess of leptininhibits hypothalamic KiSS-1 expression in pubertal mice Korean JPediatr 55337ndash343

Alessi MC Juhan-Vague I 2006 PAI-1 and the metabolic syndromelinks causes and consequences Arterioscler Thromb Vasc Biol 262200ndash2207

Barazzoni R Aleksova A Armellini I RosaCattin M Zanetti M CarriereC Giacca M Dore F Guarnieri G Sinagra G 2012Adipokines ghrelin and obesity-associated insulin resistance in

nondiabetic patients with acute coronary syndrome Obesity 202348ndash2353

Bray G Bellanger T 2006 Epidemiology trends and morbidities ofobesity and the metabolic syndrome Endocrine 29109ndash117

Burt DW 2006 Chicken genome current status and future opportuni-ties Genome Res 151692ndash1698

Burt DW Bruley C Dunn IC Jones CT Ramage A Law AS MorriceDR Paton IR Smith J Windsor D et al 1999 The dynamics ofchromosome evolution in birds and mammals Nature 402411ndash413

Campos D Palin M Bordignon V Murphy B 2008 The ldquobenefi-cialrdquo adipokines in reproduction and fertility Int J Obesity 32223ndash231

Caraty A Decourt C Briant C Beltramo M 2012 Kisspeptins and thereproductive axis potential applications to manage reproduction infarm animals Domest Anim Endocrinol 4395ndash102

Chang YH Chang DM Lin KC Shin SJ Lee YJ 2011 Visfatin in over-weightobesity type 2 diabetes mellitus insulin resistance metabolicsyndrome and cardiovascular diseases a meta-analysis and systemicreview Diabetes Metab Res Rev 27515ndash527

Chou HF Scanes CG 1988 Influence of age strain and beta-adrenergicagonist on insulin sensitivity in chicks as determined by anadaptation of the euglycemic clamp technique Poult Sci 67470ndash473

Crooijmans RPMA Dijkhof RJM Veenendaal T van der Poel JJ NichollsRD Bovenhuis H Groenen MAM 2001 The gene orders on hu-man chromosome 15 and chicken chromosome 10 reveal multipleinter- and intrachromosomal rearrangements Biol Evol 182102ndash2109

de Givry S Bouchez M Chabrier P Milan D Schiex T 2005CAR(H)(T)AGene multipopulation integrated genetic and radiationhybrid mapping Bioinformatics 211703ndash1704

Douaud M Feve K Gerus M Fillon V Bardes S Gourichon D DawsonDA Hanotte O Burke T Vignoles F et al 2008 Addition of themicrochromosome GGA25 to the chicken genome sequence as-sembly through radiation hybrid and genetic mapping BMCGenomics 9129

Dridi S Swennen Q Decuypere E Buyse J 2005 Mode of leptin action inchicken hypothalamus Brain Res 1047214ndash223

Dupont J Chen JW Derouet M Simon J Leclercq B Taouis M 1999Metabolic differences between genetically lean and fat chickens arepartly attributed to the alteration of insulin signaling in liver J Nutr1291937ndash1944

Dupont J Tesseraud S Derouet M Collin A Rideau N Crochet S GodetE Cailleau-Audouin E Metayer-Coustard S Duclos MJ et al 2008Insulin immuno-neutralization in chicken effects on insulin signal-ing and gene expression in liver and muscle Endocrinology 197531ndash542

Dupont J Tesseraud S Simon J 2009 Insulin signaling in chicken liverand muscle Gen Comp Endocrinol 16352ndash57

Dupont J Metayer-Coustard S Ji B Rame C Gespach C Voy B Simon J2012 Characterization of major elements of insulin signaling cascadein chicken adipose tissue apparent insulin refractoriness Gen CompEndocrinol 17686ndash93

Dupont J Reverchon M Bertoldo M Froment P 2014 Nutritional sig-nals and reproduction Mol Cell Endocrinol 382527ndash537

Etches RJ Petitte JN 1990 Reptilian and avian follicularhierarchiesmdashmodels for the study of ovarian development J ExpZool 4112ndash122

Fantuzzi G 2005 Adipose tissue adipokines and inflammation J AllergyClin Immunol 115911ndash919

Finucane F Luan J Wareham N Sharp S OrsquoRahilly S Balkau B FlyvbjergA Walker M Hoslashjlund K Nolan JJ et al 2009 Correlation of theleptin adiponectin ratio with measures of insulin resistance in non-diabetic individuals Diabetologia 522345ndash2349

Friedman-Einat M Cogburn LA Yosefi S Hen G Shinder D Shirak ASeroussi E 2014 Discovery and characterisation of the first genuineavian leptin gene in the rock dove (Columbia livia) Endocrinology155(9)3376ndash3384

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ovember 12 2014

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Griffin D Burt DW 2014 All chromosomes great and small 10 years onChromosome Res 221ndash6

Groenen MAM Cheng HH Bumstead N Benkel BF Briles WE Burke TBurt DW Crittenden LB Dodgson J Hillel J et al 2000 A consensuslinkage map of the chicken genome Genome Res 10137ndash147

Hajer G van Haeften T Voorrips R 2008 Adipose tissue dysfunctionin obesity diabetes and vascular diseases Eur Heart J 292959ndash2971

Hausman G Richard B Lents C 2012 Leptin and reproductive functionBiochimie 942075ndash2081

Hazelwood R Kimmel J Pollock H Barksdale B 1968 Biological charac-terization of chicken insulin activity in rats and domestic fowlEndocrinology 831331ndash1336

Hiller LW Miller W Birney E Warren W Hardison RC Ponting CP BorkP Burt DW Groenen MAM Delany ME et al 2004 Sequenceand comparative analysis of the chicken genome provide uniqueperspectives on vertebrate evolution Nature 432695ndash716

Hong YH Lillehoj HS Lee SH Park DW Lillehoj EP 2006Molecular cloning and characterization of chicken lipopolysaccha-ride-induced TNF-alpha factor (LITAF) Dev Comp Immunol 30919ndash929

Huang G Li J Wang H Lan X Wang Y 2014 Discovery of a novelfunctional leptin protein (LEP) in zebra finches evidence for theexistence of an authentic avian leptin gene predominantly expressedin the brain and pituitary Endocrinology 13en20141084

Hughes AL Friedman R 2008 Genome size reduction in the chicken hasinvolved massive loss of ancestral protein-coding genes Mol BiolEvol 252681ndash2688

Jacques C Holzenberger M Mladenovic Z Salvat C Pecchi EBerenbaum F Gosset M 2012 Proinflammatory actions of visfa-tinnicotinamide phosphoribosyltransferase (Nampt) involve regu-lation of insulin signaling pathway and nampt enzymatic activity JBiol Chem 28715100ndash15108

Johnson AL Leone EW 1985 Ovine luteinizing hormone-induced ste-roid and luteinizing-hormone secretion and ovulation in intact andpregnant mare serum gonadotropin-primed hens Poult Sci 642171ndash2179

Kaiya H Kangawa K Miyazato M 2013 Update on ghrelin biology inbirds Gen Comp Endocrinol 190170ndash175

Kanda S Oka Y 2013 Structure synthesis and phylogeny of kisspeptinand its receptor Adv Exp Med Biol 7849ndash26

Kuprash DV Alimzhanov MB Tumanov AV Grivennikov SI ShakhovAN Drutskaya LN Marino MW Turetskaya RL Anderson AORajewsky K et al 2002 Redundancy in tumor necrosis factor(TNF) and lymphotoxin (LT) signaling in vivo mice with inactivationof the entire TNFLT locus versus single-knockout mice Mol Cell Biol228626ndash8634

Kwon H Pessin JE 2013 Adipokines mediate inflammation and insulinresistance Front Endocrinol (Lausanne) 471

Landry D Cloutier F Martin LJ 2013 Implications of leptin in neuroen-docrine regulation of male reproduction Reprod Biol 131ndash14

Luque RM Kineman RD Tena-Sempere M 2007 Regulation of hypo-thalamic expression of KiSS-1 and GPR54 genes by metabolic factorsanalyses using mouse models and a cell line Endocrinology 1484601ndash4611

Mao X Kikani CK Riojas RA Langlais P Wang L Ramos FJ Fang QChrist-Roberts CY Hong JY Kim RY et al 2006 APPL1 binds toadiponectin receptors and mediates adiponectin signalling andfunction Nat Cell Biol 8516ndash523

Markov GV Paris M Bertrand S Laudet V 2008 The evolution of theligandreceptor couple a long road from comparative endocrinol-ogy to comparative genomics Mol Cell Endocrinol 2935ndash16

Meslin C Mugnier S Callebaut I Laurin M Pascal G Poupon A GoudetG Monget P 2012 Evolution of genes involved in gamete interac-tion evidence for positive selection duplications and losses in ver-tebrates PLoS One 7e44548

Morisson M Denis M Milan D Klopp C Leroux S Bardes S Pitel FVignoles F Gerus M Fillon V et al 2007 The chicken RH map cur-rent state of progress and microchromosome mapping CytogenetGenome Res 11714ndash21

Morisson M Lemiere A Bosc S Galan M Plisson-Petit F Pinton PDelcros C Feve K Pitel F Fillon V et al 2002 ChickRH6 a chickenwhole-genome radiation hybrid panel Genet Sel Evol 34521ndash533

Muffato M Louis A Poisnel CE Crollius HR 2010 Genomicus a data-base and a browser to study gene synteny in modern and ancestralgenomes Bioinformatics 261119ndash1121

Pinilla L Aguilar E Dieguez C Millar RP Tena-Sempere M 2012Kisspeptins and reproduction physiological roles and regulatorymechanisms Physiol Rev 921235ndash1316

Pitel F Faraut T Bruneau G Monget P 2010 Is there a leptin gene in thechicken genome Lessons from phylogenetics bioinformatics andgenomics Gen Comp Endocrinol 1671ndash5

Pradidarcheep W Labruyere WT Dabhoiwala NF Lamers WH 2008Lack of specificity of commercially available antisera better specifi-cations needed J Histochem Cytochem 561099ndash1111

Prokop JW Schmidt C Gasper D Duff RJ Milsted A Ohkubo T Ball HCShawkey MD Mays HL Jr Cogburn LA et al 2014 Discovery ofthe elusive leptin in birds identification of several ldquomissing linksrdquoin the evolution of leptin and its receptor PLoS One 9e92751

Saper CB 2005 An open letter to our readers on the use of antibodies JComp Neurol 493477ndash478

Sharp PJ Dunn IC Waddington D Boswell T 2008 Chicken leptin GenComp Endocrinol 1582ndash4

Simon J 1989 Chicken as a useful species for the comprehension ofinsulin action Crit Rev Poult Biol 2121ndash148

Simon J Leclercq B 1982 Longitudinal-study of adiposity in chickensselected for high or low abdominal fat-contentmdashfurther evidence ofa glucose-insulin imbalance in the fat line J Nutr 1121961ndash1973

Simon J Rideau N Taouis M 2009 Reply to viewpoints by PJ Sharp ICDunn D Waddington and T Boswell [Chicken Leptin General andComparative Endocrinology 158 2ndash4 (2008)] Gen Comp Endocrinol161159ndash159

Solinhac R Leroux S Galkina S Chazara O Feve K Vignoles F MorissonM Derjusheva S Bedrsquohom B Vignal A et al 2010 Integrative map-ping analysis of chicken microchromosome 16 organization BMCGenomics 11616

Steppan CM Wang J Whiteman EL Birnbaum MJ Lazar MA 2005Activation of SOCS-3 by resistin Mol Cell Biol 251569ndash1575

Takimoto T Sato K Akiba Y Takahashi K 2008 Role of chicken TL1A oninflammatory responses and partial characterization of its receptor JImmunol 1808327ndash8332

Taouis M Chen JW Daviaud C Dupont J Derouet M Simon J 1998Cloning the chicken leptin gene Gene 208239ndash242

Taouis M Derouet M Chevalier B Simon J 1993 Corticosterone effecton insulin-receptor number and kinase-activity in chicken muscleand liver Gen Comp Endocrinol 89167ndash175

Taouis M Dridi S Cassy S Benomar Y Raver N Rideau N Picard MWilliams J Gertler A 2001 Chicken leptin properties and actionsDomest Anim Endocrinol 21319ndash327

Taouis M Taylor SI Reitman M 1996 Cloning of the chicken insulinreceptor substrate 1 gene Gene 17851ndash55

Tena-Sempere M 2006 GPR54 and kisspeptin in reproduction HumReprod Update 12631ndash639

Tokushima Y Sulistiyanto B Takahashi K Akiba Y 2003 Insulin-glucoseinteractions characterised in newly hatched broiler chicks Br PoultSci 44746ndash751

Touchburn S Simon J Leclercq B 1981 Evidence of a glucose-insulinimbalance and effect of dietary-protein and energy-level in chickensselected for high abdominal fat-content J Nutr 111325ndash335

Trapnell C Williams BA Pertea G Mortazavi A Kwan G van Baren MJSalzberg SL Wold BJ Pachter L 2010 Transcript assembly andquantification by RNA-Seq reveals unannotated transcripts and iso-form switching during cell differentiation Nat Biotechnol 28511ndash515

Um HN Han JM Hwang JI Hong SI Vaudry H Seong JY 2010 Molecularcoevolution of kisspeptins and their receptors from fish to mam-mals Ann NY Acad Sci 120067ndash74

Voorrips RE 2002 MapChart software for the graphical presentation oflinkage maps and QTLs J Hered 9377ndash78

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ovember 12 2014

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Volker M Backstrom N Skinner BM Langley EJ Bunzey SK Ellegren HGriffin DK 2010 Copy number variation chromosome rearrange-ment and their association with recombination during avian evo-lution Genome Res 20503ndash511

Wang C Xin X Xiang R Ramos FJ Liu M Lee HJ Chen H Mao XKikani CK Liu F et al 2009 Yin-Yang regulation of adiponectinsignaling by APPL isoforms in muscle cells J Biol Chem 28431608ndash31615

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ovember 12 2014

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