Corticotropin-Releasing Hormone Family Evolution: Five Ancestral Genes Remain in Some Lineages

57:1

j c r cardoso and others Evolution of the CRH family 57:1 73–86 Research

Corticotropin-releasing hormone family evolution: five ancestral genes remain in some lineages

João C R Cardoso1, Christina A Bergqvist2, Rute C Félix1 and Dan Larhammar2 Correspondence 1Comparative Endocrinology and Integrative Biology, Centre of Marine Sciences, Universidade do should be addressed Algarve, Campus de Gambelas, Faro, Portugal to D Larhammar 2Department of Neuroscience, Science for Life Laboratory, Uppsala University, Uppsala, Sweden Email [email protected]

Abstract

The evolution of the peptide family consisting of corticotropin-releasing hormone Key Words (CRH) and the three urocortins (UCN1-3) has been puzzling due to uneven evolutionary ff CRH/UCN rates. Distinct gene duplication scenarios have been proposed in relation to the two ff phylogeny basal rounds of vertebrate genome doubling (2R) and the teleost fish-specific genome ff gene duplication doubling (3R). By analyses of sequences and chromosomal regions, including many ff synteny neighboring gene families, we show here that the vertebrate progenitor had two ff chromosome duplication peptide genes that served as the founders of separate subfamilies. Then, 2R resulted in a total of five members: one subfamily consists of CRH1, CRH2, and UCN1. The other subfamily contains UCN2 and UCN3. All five peptide genes are present in the slowly evolving genomes of the coelacanth Latimeria chalumnae (a lobe-finned fish), the spotted gar Lepisosteus oculatus (a basal ray-finned fish), and the elephant shark Journal of Molecular Endocrinology Callorhinchus milii (a cartilaginous fish). The CRH2 gene has been lost independently in placental mammals and in teleost fish, but is present in birds (except chicken), anole lizard, and the nonplacental mammals platypus and opossum. Teleost 3R resulted in an additional surviving duplicate only for crh1 in some teleosts including zebrafish crh1a( and crh1b). We have previously reported that the two vertebrate CRH/UCN receptors arose in 2R and that CRHR1 was duplicated in 3R. Thus, we can now conclude that this peptide–receptor system was quite complex in the ancestor of the jawed vertebrates with five CRH/UCN peptides and two receptors, and that crh and crhr1 were duplicated in the teleost fish tetraploidization. Journal of Molecular Endocrinology (2016) 57, 73–86

Introduction

The corticotropin-releasing hormone (CRH) family 2004, Fox & Lowry 2013). Four independent lineages members are a group of neuropeptides that are best of structurally related peptides have been described in known for their role in the regulation of the physiological vertebrates, CRH, and three urocortins, abbreviated response to stress, emotional behavior, and anxiety in UCN1, 2, and 3. Vale and coworkers were pioneers in vertebrates (Vale et al. 1981, Dunn & Berridge 1990, Koob the discovery and functional characterization of the & Heinrichs 1999, Lovejoy & Balment 1999, Gysling et al. mammalian CRH family members. CRH was isolated

http://jme.endocrinology-journals.org © 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-16-0051 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 01:32:38PM via free access

10.1530/JME-16-0051 Journal of Molecular Endocrinology the vertebrateancestor(Lovejoy&deLannoy2013 ). of theHPA/I axiswasprobablyaninnovation that arosein and energy-relatedmetabolism. Theirroleintheregulation was probablyinvolvedinthe regulationofionhomeostasis been takentosuggestthattheancestralCRH/DHpeptide regulators ofinsectdiuresisandfoodintake,thishas related peptides,theDHpeptidesareknowntobekey the functionsofearlyvertebrateandinvertebrateCRH- not yetbeenstudied.Despitethelimitedknowledgeabout their physiologicalrolesandaffinityforthereceptors has 1999) andlophotrochozoans(Mirabeau&Joly2013),but protostomes includingnematodes(Lovejoy&Balment 2013). Relatedpeptideshavebeenfoundalsoinother peptides (Lovejoy&Jahan2006, characterized homologs of the vertebrate CRH family diuretic hormones(DH31andDH44)arethebest- ( purpuratus, andthehemichordateSaccoglossuskowalevskii Branchiostoma floridae,theechinodermStrongylocentrotus ( the urochordatesCionaintestinalisandsavignyi also beenidentifiedininvertebratedeuterostomes,namely are presentthroughoutvertebrates.ACRHhomologhas ( ( milii) the holocephalanelephantshark(Callorhinchus representatives ofbasalvertebratelineages,suchas vertebrate cladesandtheiridentificationinextant ( response (Jain stress (Coste 1996, processes includingappetitesuppression(Spina have illuminatedtheirfunctionsinotherphysiological of CRHfamilypeptidestriggeredfurtherstudiesthat interrenal (HPA/I) axis.Thewidespreaddistribution in organizingthehypothalamic–pituitary–adrenal/ andplaysacentralrole secretion fromthepituitary stimulator ofadrenocorticotropichormone(ACTH) mammalian nucleotidedatabases. mammals) (Lewis (38 aainmammals)(Reyes amphibian sauvagine(Vaughan was concludedtobetheorthologoffishurotensinIand (40 aainmammals)wasobtainedfromratmidbrainand (aa) inmammals(Vale from sheephypothalamus and consistsof41aminoacids ). In protostomes, the arthropod Mirabeau &Joly2013).Inprotostomes,thearthropod Lovejoy &Barsyte-Lovejoy2010),thecephalochordate Petromyzon marinus) (Roberts Nock Imperatore DOI: 10.1530/JME-16-0051 http://jme.endocrinology-journals.org Research Research CRH family members have been described in many CRH familymembershavebeendescribedinmany Studies inmammalsrevealedthatCRHisthemajor Contarino ) and the agnathan sea lamprey al.2011)andtheagnathansealamprey et et al. 2006, ), regulation of the inflammatory . 2000),regulationoftheinflammatory et al et al. 1991),andreproductioninvertebrates et al. 2000),cardiovascularadaptationto . 2001)wereretrievedbysearching et al . 1981).Subsequently, UCN1 et al Tao et al. 2001)andUCN3(38aain et al. 2007). ), shows that they . 2014),showsthatthey et al . 1995).BothUCN2 et al j

cardoso Printed inGreatBritain andothers t al. et

analysis didnotconsiderthe CRH-likepeptidepreviously in theirstudy, (Figure 4 inHwang resulting inthefourpeptide genesthatwereincluded ineachparalogon but onlytwopeptidegenes survived chromosome wasquadrupledinthetetraploidizations, ended uponseparatechromosomes,whereuponeach genes arosebeforethevertebratetetraploidizationsand the twoancestralCRH/UCN1andUCN2/UCN3precursor (Jaillon teleost fishduetotheirthirdtetraploidization(3R) Putnam two tetraploidizations(1Rand2R)(Nakatani members ineachparalogonasaconsequenceofthe Vertebrate paralogonstypicallyhavefourchromosome regions, usuallyresultingfromatetraploidization. paralogon isasetofrelatedchromosomesorchromosome genes intwoseparateparalogons(Hwang based onthechromosomallocationsoffourpeptide other modelputforth by Hwangandcoworkersis (Lovejoy &deLannoy2013, second tetraploidization generated theextant quartet ancestor andUCN2/UCN3ancestor. Subsequently, the tetraploidization event,resultingintheCRH/UCN1 single ancestralgenewasduplicatedinthefirst The model by Lovejoy and coworkers proposed that a from onetofourCRHfamilymembersinvertebrates. have been proposed to explain the duplications leading de Lannoy2013).Two models distinctevolutionary UCN2 andUNC3(Lovejoy&Jahan 2006, consisting ofCRHandUCN1theotherharboring suggested thattwosubfamiliesexistinvertebrates,one the lowestaffinity(Gyslinget al.2004). by allfourpeptidesinmammalswithUCN3displaying their similarpharmacologicalactions.CRHR2isactivated strongly activatedbyCRHandUCN1thisexplains few species(Cardoso to Crhr1aandCrhr1b,whichwereonlyretainedina 2004) that generated a duplicate of the third roundofgenomedoublingcalled3R(Jaillon . 2008).Theancestorofteleostfishwentthrougha et al so-called 1Rand2Revents(Nakatani vertebrate genomedoubling(Cardoso that arosebygeneduplicationinthetworoundsofbasal CRHreceptors(CRHR1andCRHR2) two highlyconserved . 2014, et al coupled receptors(GPCRs)(Hwang of receptorsthatbelongtoclassBtheG-protein-

Evolution oftheCRHfamily Published byBioscientifica Ltd. Sequence comparisonsoftheCRH-relatedpeptides The CRH/DH-likepeptidesbindandactivateagroup . 2004).Hwangandcoworkerssuggestedthat et al t al.2008),andasmanyeightmembersin et Lovejoy . 2014).Vertebrates usuallyhave et al . 2014).Inmammals,CRHR1is et al Downloaded fromBioscientifica.com at09/25/202101:32:38PM Lovejoy t al.2013, et . 2007, et al 57 57 t al. 2013). Their et crhr1 gene, leading t al.2014).The et : : t al.2014),the et 1 1 . 2013).A et al . 2007 , et al Lovejoy & Cardoso Putnam . et al 74 74 via freeaccess

Journal of Molecular Endocrinology CRH systemarediscussed. implications ofthisancientancestralcomplexitythe reported (Grone&Maruska2015a).Theevolutionary that aroseintheteleosttetraploidization,asrecently . 2015),isinfactateleost-specific genecopyofCRH et al proposed tobeanancestralvertebrateduplicate(Hosono CRH-related peptideinteleostfish,namedteleocortinand synteny andparalogonanalysisthattherecentlyreported mammals andinteleostgenomes.We alsoconfirmby the majorlineages.However, CRH2waslostinplacental family peptides,allofwhicharestillpresentinsome two tetraploidizations(1Rand2R)resultedinfiveCRH before thefirsttetraploidization(1R).Subsequently, the CRH familygenesexistedinthevertebrateancestor already several neighboringgenefamilies,weconcludethattwo chromosomal regions,includingphylogeneticanalysesof and paralogoncomparisons.Aftercomparisonoflarge genesynteny,in-depth analysesofsequences,conserved receptor family(Cardoso same approachasinourrecentanalysisoftheCRH-DH investigation oftheCRH/UCNpeptidefamilyusing early-branching vertebratelineages.We describehereour that CRH2ispresentinrepresentativesfromdifferent CRH-DH receptorfamily(Cardoso vertebrate tetraploidizations. The latterreportsuggestedthatCRH2aroseinthebasal lineages and was named CRH2 (Grone & Maruska 2015b). was recentlyreportedtobepresentinothervertebrate arisen earlyinthislineage(Nock identified incartilaginousfish,initiallyproposedtohave ). gray short-tailedopossum (Monodelphisdomestica). (an earlydivergentmammalian clade)andthemarsupial anatinus) thatisamemberofthe orderMonotremata genomes, theduck-billed platypus(Ornithorhynchus ( basal sarcopterygian representativetheAfricancoelacanth ray-finned fishthespottedgar(Lepisosteusoculatus), Searches includedthegenomesofearlydiverging al.2011)peptideprecursorusingtheblastalgorithm. et deduced elephant shark (Callorhinchus ) milii CRH2 (Nock and UCN3 (NP_444277) peptide precursors and the (NP_000747), UCN1(NP_003344),UCN2(NP_149976), in severalvertebrategenomeswiththehumanCRH Searches forCRHfamilymemberswereperformed Database miningandsequenceretrieval Methods ), and also two mammalian key Latimeria chalumnae),andalsotwomammalian key http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0051 Research During thecourseofourpreviousanalyses t al.2014),bycombining et j et al. 2011).Thispeptide

et al. 2014),wetoofound c

cardoso Printed inGreatBritain andothers

Criterion (AIC)statisticalmodel (Abascal protein phylogenyaccording totheAkaikeInformation submitted toProtTest 2.4toselectthebestmodelstudy peptide sequencesproduced inClustalWthatwas were constructedbasedon the alignmentof78mature to assignaccuracytheclades(Felsenstein1985).Trees and neighbor-joining(NJ)methodsbootstrapped trees werebuiltusingthemaximumlikelihood(ML) GeneDoc (http://iubio.bio.indiana.edu/).Phylogenetic (http://www.genome.jp/tools/clustalw/) andeditedin compared across species and were aligned with ClustalW The deducedpeptideprecursorsandmaturepeptideswere Sequence alignmentsandphylogeneticanalysis data givenattheendofthisarticle). Data1,seesectiononsupplementary (Supplementary 2006) toretrievethematurepeptidesforfurtheranalysis animal.uiuc.edu/cgi-bin/neuropred.py) (Southey identified orpredictedusing NeuroPred(http://stagbeetle. proteolytic consensuscleavagesites(RR,KR,KK)were precursor. Putativemonobasic,dibasic,ortribasicpeptide org/translate/) to obtain the most complete peptide Expasy nucleotidetranslatingtool(http://web.expasy. sequence hit and then translated into protein using the extending 1 sequences wereretrievedfromthegenomeassemblies motifs werefoundinnonannotatedgenomeregions, identify theproteinfamilymotifs.Whenshortsequence and alsointhePfamdatabase(http://pfam.xfam.org)to orthologs availableatintheNCBIdatabaseusingBLASTp identity wasconfirmedbysearching againstthehuman additional CRHfamilymembers. esharkgenome.imcb.a-star.edu.sg ) wasalsoqueriedforany fish theelephantshark(Callorhinchusmilii)http:// jlampreygenome.imcb.a-star.edu.sg/ ). Thecartilaginous and theArctic lamprey(Lethenteron camtschaticum)(http:// lamprey (Petromyzon marinus)(http://www.ensembl.org/) uchile.cl). Searches wereextendedtotwolampreys,thesea from theSalmonDBdatabase(http://salmondb.cmm. the teleostAtlanticsalmon(Salmonsalar)thatisavailable the ENSEMBLdatabase(http://www.ensembl.org/ ) andalso ( latipes),three-spinedstickleback rerio), medaka(Oryzias Nile tilapia(Oreochromis niloticus),zebrafish(Danio the teleostfishesJapanesepufferfish(Takifugu ), rubripes the amphibianXenopustropicalis ( chicken (Gallusgallus),theanolelizardAnoliscarolinensis ), Other vertebrategenomeswerealsoanalyzedincluding

Gasterosteus aculeatus),andAtlanticcod(Gadusmorhua)in Evolution oftheCRHfamily Published byBioscientifica Ltd. The predictedpeptideprecursorswereretrievedand Mb upstreamanddownstreamofthe Downloaded fromBioscientifica.com at09/25/202101:32:38PM Xenopus tropicalis ) and 57 : 1 t al.2005). et t al. et 75 via freeaccess

Journal of Molecular Endocrinology DOI: 10.1530/JME-16-0051 http://jme.endocrinology-journals.org Research Table 1 Accession numbers and origin of the CRH/UCN sequences retrieved. Research

CRH1 CRH2 UCN1 UCN2 UCN3 CRH/UCN1 UCN2/UCN3 TETRAPOD Human NP_000747 ni NP_003344 NP_149976 NP_444277 Chr 8_66.176 Chr 2_27.307 Chr3_48.5 Chr10_5.36 Opossum ENSMODP00000036360 na ENSMODP00000028973 na ENSMODP00000023062 Chr 3_171 Chr 1_472 Chr 1_508.59 Chr 6_194 Chr8_121.44 Platypus ENSOANP00000003650 Ultra337_3 ni na na Chr 7_1.6 Ultra 32_0.7 Contig13_0.6 ENSMODP00000023062 Chr 8_121 Chicken ENSGALP00000024980 ni ni na AGC65587.1

Chr 2_114 Chr 12_9 Chr1_1.16 j

Chr 4_30 Chr 4_156 Chr5_3.75 cardoso Xenopus ENSXETP00000058657 ni ni ni ENSXETP00000035550

Printed inGreatBritain GL173273.1_0.555 GL172647.1_1.43 andothers LOBE-FINNED FISH Coelacanth ENSLACP00000002936 na ENSLACP00000011213 na na JH128518.1_0.54557 JH129487.1_0.06 JH127070.1_0.454 JH126690.1_1 JH126940.1_1.33 RAY-FINNED FISH Spotted gar ENSLOCP00000021917 na ENSLOCP00000022093 na ENSLOCP00000022005 LG 9_6 LG 18_11 LG1_17.175 LG 5_51 LG8_11.31 Tilapia a-ENSONIP00000025716 ni ENSONIP00000026599 GL831152.1_4 ENSONIP00000025826

Evolution oftheCRHfamily

Published byBioscientifica Ltd. GL831416.1_0. 3789 GL831530.1_0.242 GL831167.1_2.25 b-ENSONIP00000025725 GL831157.1_0.8489 Fugu b-ENSTRUP00000022019 ni ENSTRUP00000030463 na ENSTRUP00000040554 scaffold 95_0.421 Scaffold 120_0.061 Scaffold 116_0.09 Scaffold 8_1.19 Zebrafish a-ENSDARP00000118835 ni NP_001025351 ni NP_001076423 Chr 2_42.21 Chr_20_38.749 Chr4_15.05 b-NP_001007380 Chr 24_25.45 Stickleback a-ENSGACP00000003885 ni ENSGACP00000008094 na ENSGACP00000026067

Downloaded fromBioscientifica.com at09/25/202101:32:38PM groupXXI_7306566 groupXVIII_3309909 groupXVII_9846005 groupIV_29552483 b-na scaffold_120_91380 Cod a-ENSGMOG00000020152 ni ENSGMOP00000017865 ENSGMOP00000021625 ENSGMOT00000013071* GeneScaffold_378_17550 GeneScaf- GeneScaffold_904_22574 GeneScaf- b-ENSGMOT00000002317* fold_2370_28022 fold_1619_113273 57 GeneScaffold_2511_7808 57 : : 1 Medaka a-NP_001121990 ni NP_001295911.1 NP_001121991.1 NP_001121992.1 1 Chr20_15301114 Chr24_260595 Chr 5_31842935 Chr23_15300487 b-XP_011484530 Chr17_10775330 Salmon a1-NP_001135062.1 ni AGKD01034474.1 a1-AGKD01002194.1 AGKD01004252.1 a2-XP_014036838.1 a2-AGKD01007057.1 76 76 b1-ACM09696.1 via freeaccess b2-XP_014045819.1 Journal of Molecular Endocrinology http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0051 Smelt a-ACO09991.1 un un un un Research b-ACO10011.1 JAWLESS FISH Elephant SINCAMP00000023414 AAVX01072774.1 AAVX01644813.1 SINCAMP00000005227 SINCAMP00000001034 shark Scaffold 48_2.54 Scaffold 6_0.566 Scaffold 124_1.27 Scaffold 15_4.10 Scaffold 17_1.89 AGNATHA Arctic KE994103.1 KE993813.1 lamprey Scaf- Scaf- fold00432_0.062 fold00142_0.52 KE993984.1 Scaf- fold00313_0.221 KE993827.1 j

Scaf- c

fold00156_1.151 cardoso Sea na na lamprey GL480439_0.0003 GL476347_0.32 Printed inGreatBritain andothers

Accession numbers and positions in the genome (Mb) and chromosome locations (in italics) of the vertebrate CRH family genes. Na, not available; ni, not identified. *indicates the sequences that were annotated in the database as pseudogenes. For guidance, CRH and UCN a and b paralogs are indicated before the accession numbers. CRH2 gene. the ‘standard’CRHgenetodistinguishitfrom thenovel and teleosts.Forclarity, wewillusethenameCRH1for other vertebratelineagesincluding placentalmammals in platypusandopossum(see below),butismissingin and ispresentalsointheanolelizardprobably orthologous betweencoelacanth,gar, andelephantshark, al.2011 , et UCN2, andUCN3peptides.TheCRH2peptide(Nock four oftheseasorthologsmammalianCRH,UCN1, and chromosomalanalysesdescribedbelowidentified spotted gar, andelephantshark.Extensivephylogenetic diverged earlyinvertebrateevolution,namelycoelacanth, retrieved from three species representing lineages that 1.Atotaloffivedistinctsequenceswere listed inTable genome databasesidentified alargenumber ofgenesas Searches forCRHfamily membersinseveralvertebrate CRH genefamilymembersinvertebrates Results for theCRHfamilymembers. analyses followingasimilarprocedureasdescribedabove (ortholog vsparalog)wasinvestigatedbyphylogenetic assemblies forthedifferentspecies,andgeneidentity thegenome Orthologous genesweresoughtbyquerying oxytocin/vasopressin receptorgenes(Lagman 2010, the opioidreceptorandpeptidegenes(Sundstrom selected basedonpreviousparalogonannotationsof and elephantsharkgenomes.Candidategeneswere genes wereidentifiedinthehuman,chicken,zebrafish, CRH familygeneswerecharacterizedandorthologous The chromosomalenvironmentsofthefivespottedgar Gene syntenycomparisons with MLandNJanalysissharedsimilartopologies. 1000 bootstrapreplicates.Theconsensus trees obtained deletion and model and fixed G (1.528) with pairwise version 6(Tamura method (Saitou&Nei1987)usingtheprogramMEGA replicates. SequencedatawerealsoanalyzedwiththeNJ for internalbranchingwasassessedusing100bootstrap and fixedgammashapeparameter(1.229).Reliability following parameters:4gammadistributedratecategories . 2010)withtheLGsubstitutionmodeland et al ATGC (http://www.atgc-montpellier.fr/phyml/) (Guindon The MLtreewasbuiltwithPhyML3.0availablefrom

Evolution oftheCRHfamily Published byBioscientifica Ltd. Larhammar Grone &Maruska2015a)wasfoundtobe t al.2015)andthevisualopsin et . 2013)withtheJTTsubstitution et al Downloaded fromBioscientifica.com at09/25/202101:32:38PM 57 : 1 et al. 2013). . et al 77 via freeaccess Journal of Molecular Endocrinology are 100%identicalatnucleotide level,suggestingthat to belocalizedintandem onscaffoldGL173273that too. InX.tropicalis, twoCRH1genecopiesarepredicted apply also to the lizard as it has microchromosomes Sundstrom the opioidpeptideandreceptorgenes(Dreborg analyzed indetailbyus,namelytheparalogonthatcontains related chromosome regions), whichhave been previously CRH family genes are located in two paralogons (sets of the same chromosomal regions. This showed that the identified andanalyzedgenefamilieswithmembersin (Lovell known tobeunderrepresentedinthegenomeassemblies chr_20_8.814 Mb).Themicrochromosomesofchickenare JH603360_1.093 KB744325_0.354 in otherbirdgenomessuchasduck(Anasplatyrhynchos , for chickenbecauseputativeCRH2geneswereidentified in theassembly. Thispossibilityappearsespecially likely genomes buthavenotyetbeensequencedandincluded possible thatthemissinggenesareindeedpresentin The frog, finally, lacks UCN2. Chickenhas,butlacksCRH2andUCN1 also hastheCRH2geneandUCN1gene,butlacks contain thefamilymembersCRH1andUCN3.Thelizard The genomesofchicken,anolelizard,andX.tropicalis Nonmammalian tetrapods genomes ofplacentalmammals. 1).Thisgeneisabsentfromthe Chr1_472Mb (Table on chromosome Ultra337_3Mb and in opossum on mammals haveaputativeCRH2genelocatedinplatypus not befoundinplatypus.Bothofthesenonplacental UCN3 genes. Opossum also had tailed opossum,werefoundtohaveCRH1, mammals, theduck-billedplatypusandgrayshort- family membersexist.Thegenomesoftwononplacental In humanandotherplacentalmammals,fourCRH Mammals teleost tetraploidization,seebelow). tetraploidizations (withoneadditionalcopyarisinginthe duplications tookplaceaspartofthebasalvertebrate CRH familygenesandtodeducethatthefami allowed ustodistinguishorthologsandparalogsforthe and vasopressinreceptorgenes(Lagman one that contains the visual opsin genes and the oxytoc DOI: 10.1530/JME-16-0051 http://jme.endocrinology-journals.org Research Research For eachoftheCRHfamilymembers,wealso t al. 2014, et t al.2010, et Mb), andzebrafinch(Taeniopygia guttata, Mb), flycatcher(Ficedulaalbicollis, 2015, CRH2, Larhammar Hron UCN1, and UCN1, but this gene could j t al. 2015). This might et

cardoso t al.2015)andthe et UCN2. It remains . 2013).This et al Printed inGreatBritain andothers UCN2, and et al. 2008, in ly

Only crhb, asrecentlydonealsobyGroneandMaruska(2015a). teleost 3Revent;hence,wehavenamedthemcrhaand (see below)showedthatthesetwocopiesderivefromthe and cod. Phylogenetic analyses and synteny comparisons investigated includingzebrafish,stickleback, medaka, Two Ray-finned fish recent duplication. assembly ratherthantheresultofaspecies-specific they maybeaconsequenceofanerrorinthegenome makes itmorelikelythattwo genesaremissinginthe each otheronly10–30 genes, therecentdivergence ofthesetwospeciesfrom possible thatP. marinusmayhavelosttwoofthepepti found intheformerandfour inthelatter. Althoughitis Lethenteron camtschaticum.Two CRHfamilygeneswere lamprey were foundinthegenomesoftwolampreys,sea identified, CRHR1(Cardosoet al.2014). more intriguingthatsofaronlyonereceptorhasbeen CRH familypeptidesintheelephantsharkmakesiteven ). Thepresenceofthefullset and coelacanth(Table 1 fish displaysthesamegenerepertoireasspotted gar that alsotheUCN2geneexists.Thus,thiscartilaginous UCN3, and the novel CRH2. Our investigation revealed identified intheelephantshark,namelyCRH1,UCN1, Genes forfourCRHfamilymembershavebeenpreviously Elephant sharkandlampreys and thecoelacanth,alobe-finnedfish(Table 1). the spottedgarthatdivergedbeforeteleostradiation family members,includingCrh2,wereidentifiedbothin lack anorthologofthenovelcrh2gene.AllfiveCRH & Maruska2015a).Alloftheteleostgenomesanalyzed encode identicalmatureCrhaandCrhbpeptides(Grone crha2; and the extragenomedoubling,asecondcopyofbothcrha specific fourthtetraploidization(4R).Asaconsequenceof received asecondcopy, presumablyinthesalmonid- of zebrafish that has none and Atlantic salmon that found inasinglecopytheteleostswithexception duplicates have been lost. Likewise, the ucn2gene was investigated, implyingthatthe3Rtetraploidization copies ofucn1anducn3geneswerefoundinallteleosts

Evolution oftheCRHfamily Published byBioscientifica Ltd. Homologs ofthebonyvertebrateCRH/UCNmembers crhb were gained in the Atlantic salmon (crha1 and crh1 geneswerefoundinmostoftheteleosts crhb was found in the crhb1 and Petromyzon marinusandtheArctic lamprey crhb2). These presumed 4R duplicates Mya (Kuraku&Kuratani2006 ) Downloaded fromBioscientifica.com at09/25/202101:32:38PM Takifugu genome. Single 57 57 : : 1 1 78 78 de via freeaccess

Journal of Molecular Endocrinology and theL-T-F-H-L/I-L-R motifafteritarealsoconserved. CRH2/UCN1 subfamily, thePbeforeS-L/I-Dmotif acrossthefourspecies.Withinalso conserved theCRH1/ terminus andtheAlocated threepositionsbeforeare spotted gar, andelephantshark.TheN nearthecarboxy- betweenhuman, coelacanth, peptides andconserved The S-L/I-DmotifattheN-terminusispresentinallfive 1). CRH1/CRH2/UCN1 andUCN2/UCN3clades(Fig. common toallfamilymembersorcharacteristicforthe Specific aminoacidmotifswereidentifiedthat are CRH/UCN peptides Sequence comparisonofthevertebrate synteny isrequiredtoconfirmorthology. vertebrates; however, moredetailedanalysisofgene genomes possessasimilargenecontenttotheother in thesealampreygenomessuggestingthat and UCN2seemtoexistintheArctic lampreyandUCN2 gnathostome peptideclades,andputativeCRH1,CRH2, Some of the agnatha sequences tend to cluster with the P. marinusgenomeassemblyduetoincompletecoverage. http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0051 Research j

cardoso Printed inGreatBritain andothers selection pressure(shortertree branches)thantheother presumably havebeensubjected tostrongerconservative The peptidesCRH1andUCN3 evolvemoreslowlyand pressures. the tree branches suggest distinct evolutionary have lowbootstrapsupport andthevariablelengthsof each peptidecluster, thein-grouprelationshipsgenerally form aseparateclusterwithintheCRH1clade.Within The newlyidentifiedCRH2sequencesingnathostomes 2). other comprising the UCN2/UCN3 subfamily (Fig. that containstheCRH1/CRH2/UCN1subfamilyand of twomajorandclearlyseparatepeptideclusters,one branch topologies.Bothphylogenetictreesarecomposed Fig. 2BNJ (Supplementary ) methodsthatproducedsimilar trees withbothML(Fig. 2 Fig. 2A andSupplementary ) and Fig. 1(Supplementary ) wereusedtoconstructevolutionary The maturepeptidesequencesoftheCRHfamilymembers Phylogeny oftheCRHfamilymembers motifswerealsoidentified(Fig. 1). several conserved between CRH1andCRH2.IntheUCN2/UCN3subfamily, Five otheraminoacidswerefoundtobetotallyconserved

Evolution oftheCRHfamily Published byBioscientifica Ltd. org/10.1530/JME-16-0051. this figureisavailableathttp://dx.doi. nonconserved region.Afullcolourversionof partial conservationand,gapsindicate total conservation,smallcaselettersindicate annotated inbold:uppercaselettersrepresent each subfamilyisrepresentedbelowand and CRH2.Theconsensuspeptidesequencefor Positions shownwith‘.’arecommontoCRH1 and UCN2/UCN3subfamilies,respectively. acids conservedwithintheCRH1/CRH2/UCN1 shown markedwith‘:’or“#”aretheamino peptides areannotatedwith‘*’.Positions acids thathavebeenmaintainedacrossall cleavage sites(SupplementaryData1).Amino were identifiedbasedonconsensusproteolytic spotted gar, andelephantshark.Thepeptides family maturepeptidesinhuman,coelacanth, Sequence alignmentofthefivededucedCRH Figure 1 Downloaded fromBioscientifica.com at09/25/202101:32:38PM 57 : 1 79 via freeaccess Journal of Molecular Endocrinology (Dreborg previously describedopioid peptideandreceptorgenes CRH2, andUCN1resideinthesameparalogon asthe that they are located in two distinct paralogons: The chromosomallocations of theCRHfamilygenesshow Analyses ofneighboringgenefamilies divergence ofcyclostomesandgnathostomes. clade, showingthatthetwosubfamiliesarosebefore either the CRH1/CRH2/UCN1 clade or the UCN2/UCN3 and rate differences. The lamprey peptides group with species trajectoriesasaresultoflineage-specificevents the sequencesdonotradiateaccordingtoestablished three members. Moreover, within each peptide cluster, DOI: 10.1530/JME-16-0051 http://jme.endocrinology-journals.org Research Research . 2008, et al Sundstrom j

. 2010, et al c

cardoso Printed inGreatBritain andothers Larhammar CRH1, subfamilies). Thegenefamilies wereinvestigatedindetail subfamilies), STMN, NCOAXKRHCK/LCK/BLK/LYN , GATA , namely in eitheroftheparalogons are showninFig. 3 families thataresyntenicwith theCRHfamilymembers human, chicken, and spotted gar. Eight of these gene several neighboringgenefamiliesinthegenomes of ofgenesynteny wasfound for Extensive conservation . , 2010 et al within eachparalogon(Dreborg gene familiesrepresentedon2–4ofthechromosomes vasopressin receptorgenes(Lagmanet al.2013)Fig. 3). the paralogon harboring the visual opsin and oxytocin/ et al. 2015),andtheUCN2UCN3genesarelocatedin

Evolution oftheCRHfamily Published byBioscientifica Ltd. Each ofthesetwoparalogonshasnumerousother Lagman CAMK1, dx.doi.org/10.1530/JME-16-0051. colour versionofthisfigureisavailableathttp:// duplicates produceidenticalpeptides.Afull salmon Crha1andCrhb1arerepresentedastheir UCN3 ingreen.OnlythesequencesofAtlantic with colors:CRH1/CRH2/UCN1inpinkandUCN2/ ancestral peptidecladesarecircledandannotated The peptidesubfamiliesdescendantsofthetwo from NCBI(Crha,ACO09991.1;CrhbACO10011.1). (Osmerus mordax)Crhsequenceswereobtained is showninSupplementaryFig. 1.Thesmelt are showninTable 1 andthesequencealignment (ML) method.Accessionnumbersofthesequences peptide precursors)withthemaximumlikelihood sequences (themostconservedregionsofthe was constructedusingthematurepeptide members invertebrates.Thephylogenetictree Phylogenetic relationshipoftheCRHfamily Figure 2 . , 2013 et al Downloaded fromBioscientifica.com at09/25/202101:32:38PM PLXNA, andCACNA2D(two Larhammar t al.2008, et 57 57 : : 1 1 . ). 2015 et al Sundstrom (two 80 80 via freeaccess Journal of Molecular Endocrinology STMN, stathmin;XKRXKellbloodgroupprecursor-related family. Afullcolourversion ofthisfigureisavailableathttp://dx.doi.org/10.1530/JME-16-0051. GATA stand for:CACNA2D,voltage-dependentcalciumchannel subunitalpha2deltafamily;CAMK/PCNK,Ca2+ aligned tohighlightthesimilaritiesboth betweenspeciesandthechromosomeswithineachspecies, i.e.paralogon.Thegeneabbreviations positions (Mb)aregivenbeloweach gene.Theorderofthegenesonchromosomeshasbeenreshuffled andmembersofthesame genefamilywere five different vertebratecladesareshown.Thenumbersto the leftandrightofchromosomelinesrefertoorscaffold number. Gene Gene syntenyanalysisoftheCRH1/CRH2/UCN1andUCN2/UCN3paralogons.ChromosomallocationsCRHfamilymembersneighboring genesin Figure 3 http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0051 Research , GATA transcriptionfactorfamily;HCK/LCK/BLK/LYN , tyrosineproteinkinases;NCOAnuclearreceptorcoactivatorfamily; PLXNA,plexinAfamily; Chr2 Chr2 Chr2 Chr1 Chr1 Chr1 Chr2 Chr1 Chr6 4 0 7 9 6 3 1 stmn stmn stmn stmn stmn 34.8 12.5 1 18.2 58.4 6 . 6 4 4 1 2 3 4 0 6 2 5 Chr1 Chr8 Chr8 Chr2 Chr1 Chr2 LG9 LG1 LG6 LG18 LG11 Chr2 Chr3 Chr2 Chr2 stmn stmn 37.06 33.02 8 q p 0 3 0 1 2 c o S Sc48:4.51 Sc6:1.80 n c STMN2 STMN4 STMN1 STMN3 STMN2 STMN4 STMN1 STMN3 t 3 STMN2 STMN4 STMN1 STMN3 STMN2 STMN4 STMN1 STMN3 i 79.61 63.64 2 2 6 g 1 11.7 2.66 6 1 3.20 11 120.50 5 7 ncoa2 ncoa1 ncoa3 1 : 5 19.08 3 9 . 0 0 0 . . 2 4 8 6 8 2 . . 4 . 1 7 0 . 3 . 0 8 4 1 7 . 3 8 7 0 9 5 4 1 0 9 Sc137:0.60 Sc7:1.37 Opioid NCOA NCOA NCOA NCOA NCOA 70.1 24.49 47.5 NCOA NCOA NCOA NCOA NCOA NCOA 105.1 116.2 17.69 58 8.50 j 6.20 .

8.98 xkr7 5 c 15 03 3 2 3 2 1 3

Sc124:0.13 cardoso Sc6:10.6 Sc17:9.41 55.1 31.97 10.9 XKR4 XKR6 XKR7 XKR4 XKR6 XKR7 1 16.0 11.1 10.4 XKR4 XKR6 XKR7 paralogo XKR6 XKR7 0 18.87 49.2 31.1 .9 5.52 xkr4 xkr6 xkr6 xkr7 6 . 0 09 2 5 01 5 4 7 1 9 0 S S Printed inGreatBritain Sc6:10.55 Sc17:9.6 c c 1 1 andothers 3 1 110.8 2 0 HC LY 45.5 BLK LC HC 11.9 LYN BLK LC 2 1 5.88 5.53 3 2.05 1 HC 6 5.09 27.7 1 LYN 25.1 BLK HC LC 06.8 LYN BLK LC 0.45 . . 7.58 3 . : : 9 N hck 2 4 K bl ly 3 lc lc K K 0 0 K . K . 5 3 K K 0 K . . 2 n k k k 8 1 1 0 0 3 5 3 3 6 1S 8 1 n S Sc48:2.54 c Sc6:0.57 1 UCN1 CRH1 CRH2 27.3 66.1 UCN1 CRH1 2 114.71 10.1 UCN1 CRH1 CRH2 CRH1 25.4 42.2 6.77 38.75 4 ucn1 crha crhb 7.18 : 1 1 8 . 21 2 50 1 7 Sc:JH59141 Elephant s Sc6:8.40 c GATA GATA GATA c89:1.55 GATA5 GATA6 GATA4 J 62.4 22.1 11.6 Spoe s GATA GATA GATA 1 H GATA6 GATA4 GATA5 102.3 53.5 gata gata gata c 0 8 7.45 0 5 2.00 3 Zebraﬁsh . . 6 9 4 5 3 Chicken . 6 7 8 1 5 Human 4 7 5 6 4 5 9 6 4 5 4 6 4 5 9 0 1 3 3 dg shark Sc17:1.8 S

15.0 c UCN3 UCN2 ucn3 Evolution oftheCRHfamily 4 UCN2 UCN3 9.09 1 11.3 5 UCN3 UCN2 UCN3 UCN2 1 5.36 8 . Published byBioscientifica Ltd. 1 ar 5 1 . . 4 : 6 3 5 4 5 2 5 . 1 0 9 Sc1427:0.01 Sc191:0.78 Sc15:5.2 26.3 GATA GATA gata gata 128.48 GATA GATA GATA 8.00 GATA GATA GATA GATA GATA GATA 9.19 4.29 51.1 1 8.05 2.78 48.7 3 . 4 3 1 1 3 2 9 46 1 2 3 3 1 2 3 1 2 91 67 Opsin Sc191:0.09 Sc471.0.01 CAMK1G Sc70:3.4 CAMK1G CAMK1D CAM1D CAMK1 CAMK1D CAMK1G CAMK1D CAMK1G 4 2 gata gata gata CAMK 209.58 11.4 CAMK1 CAMK1 3 12.3 6.26 3.05 PCNK 0 4 32.7 14.6 25.7 9.76 PCNK 53.67 . 1.86 . . 9 7 1 1 2 4 3 2 2 69 5 9 01 94 1 2S camk1d camk1d camk1g camk1g paralogo PLXNA4 PLXNA2 PLXNA1 camk camk Sc15:4.3 Sc17:0.9 46.2 39.7 PLXNA3 PLXNA2 PLXNA1 PLXNA4 PLXNA4 PLXNA3 PLXNA2 PLXNA1 PLXNA4 PLXNA2 PLXNA1 132.1 pcnk 7.07 1.03 4.12 9.69 129.99 8 208.0 154.4 c70:3.68 30.6 2.70 2.23 . 1.90 2 .8 1.94 5.29 5 81 0 9 /calmodulin-dependent protein kinase family; /calmodulin-dependent proteinkinase family; 1 1 6 2 2 6 4 3 plxna4 plxna3 plxna2 CACNA2D1 CACNA2D3 32.7 CACNA2D1 14.2 Sc17:5.1 CACNA2D3 S CACNA2D1 CACNA2D3 CACNA2D1 CACNA2D3 9,76 1 c 81.9 5 7.31 4 1 0 9 4 8 5 . . . 9 0 87 . 1 : 1 4 9 Downloaded fromBioscientifica.com at09/25/202101:32:38PM 4 n 1 2 5 0 . 1 0 2S plxna1 plxna1 34.62 CACNA2D4 CACNA2D2 CACNA2D4 CACNA2D2 S CACNA2D4 CACNA2D2 CACNA2D4 CACNA2D2 6 c c63:0.03 .5 5 1.44 4 4 1 1.79 1 0 9 0 34 5 . . 1 . . : 3 6 1 8 1 6 1 . 2 cacna2d1 cacna2d1 cacna2d3 cacna2d3 1 55.9 3 9.19 8 7 Chr1 Chr2 Chr1 . . Chr Chr Chr Chr12 Chr Chr10 LG LG LG LG 7 5 0 3 3 8 1 3 5 X 1 3 7 6 2 cacna2d4 cacna2d4 cacna2d2 cacna2d2 57 5 1 1 9.41 2 9 9 . . . 5 2 1 1 : 1 Chr18 Chr25 Chr Chr Chr22 Chr23 Chr11 Chr 4 8 6 81 via freeaccess Journal of Molecular Endocrinology subfamilies andtheeight neighboringgenefamilies GATA2 werealsoduplicatedinteleost 3R. shark scaffold 6 together with For instance,thenewCRH2genemapstoelephant of theneighboringgenesfoundinothervertebrates. family genes arelocated on scaffolds that harbor at least one allowed assignmentofchromosomes,butallfiveCRH is presentinotherteleostfishgenomes. Ucn2 couldnotbeidentifiedinthezebrafishgenomebut CRH2 seems to havebeenlostin the teleostancestor. duplicateafterthe3Rtetraploidization. to asurviving above, note that for the CRH family only CRH1gave rise camk1 familyisclosewithsevenmembers.Asmentioned has becomeacompleteoctetinzebrafishafter3R,butthe the fourgenefamiliesthatwerecompletequartetsafter2R with thespottedgarandtetrapodancestor. Noneof found multiple syntenies reflecting thecommon ancestry Combining informationfromafewteleostspecies,we teleost 3R,genelossesandchromosomalrearrangements. synteny, butasusualhasamorecomplexsituationdueto from acommonancestralchromosome. support thatthesefourchromosomeregionsresulted close proximitytoCRH1/CRH2/UCN1providesfurther families havelesswell-resolvedphylogenies,buttheir 4.These Fig. DNAJC5, areincludedinSupplementary Two additionalneighboringgenefamilies,TRIMand from 2R:STMN, 3R events.Fourofthegenefamiliesarecompletequartets members thatcomplywithduplicationsinthe2Rand tree topologies and species distribution of the family 3.Allofthesegenefamiliesdisplay Fig. Supplementary for the CRH family. Phylogenetic trees are shown in with alignmentsandphylogeneticanalysesasdescribed GATA a different chromosome. Subsequently, both of the gene, andthattheduplicatedregionsweretranslocated to region containingbothaCRHfamilygeneandGATA aduplication of achromosomal the vertebratesunderwent Fig. 36 (Supplementary ). Thissuggeststhattheancestorof GATA1, 2,and3 andtheothercontainingGATA 4,5,and too formstwodistinctsubfamilies,oneconsisting of . 2013).OurphylogeneticanalysisshowsthatGATA et al to GATA family membersasnotedpreviously(Hwang spotted gar. syntenywithCRH2in thereby displayingconserved DOI: 10.1530/JME-16-0051 http://jme.endocrinology-journals.org Research Research Taken together, theduplications ofthetwoCRH The elephantsharkgenomeassemblyhasnotyet Also, thezebrafishdisplaysextensiveconserved Interestingly, bothoftheCRHsubfamiliesare linked pairs were duplicated in 2R, and HCK/LCK/BLK/LYN, GATA5, j

CAMK1, andPLXNA. cardoso CRH1, HCK, and Printed inGreatBritain andothers GATA1 , and STMN3, CRH-

and thevisualopsingene family (Lagman duplications (Fig. 3). can mostparsimoniouslybeexplainedbychromosome chromosomal regions (Dreborg as severaladditionalgenefamilies,locatedinthese (two ofwhichconsisttwosubfamilies),aswell 2008, opioid peptideandreceptor genefamilies(Dreborg in connectionwithourstudies oftheevolution investigated ingreatdetailpreviouslyourlaboratory Incidentally, thesechromosomalregions havebeen for theCRHfamilymembersbetweenthesespecies. ofsynteny allowed ustoidentifyextensiveconservation coelacanth, spotted gar, andzebrafish. Thisapproach of theirgenesinseveralspecies,primarilyhuman,African duplications, weinvestigatedthechromosomalregions ( agreement witharecentlypublishedindependentstudy 4),in tetrapods, coelacanth,andray-finnedfishes(Fig. cartilaginous fishesdivergedfromthelineageleadingto arose inthebasalvertebratetetraploidizationsbefore 3),showsthatit with itschromosomallocation(Fig. 1), along gene also in coelacanth and spotted gar (Fig. to haveariseninthislineage.Ouridentificationof identified inashark(Nock related toCRH1andUCN1.Thispeptidewaspreviously UCN2, andUCN3anadditionalpeptidemostclosely 2):thepreviouslydescribedCRH1,UCN1, clades (Fig. the gnathostomeCRHfamilymembersformfivedistinct analysis ofthepredictedmaturepeptidesrevealedthat in thegenomesofseveralvertebrates.Phylogenetic & Maruska2015b). vertebrate memberofthisfamily(Nock the recentlydiscoveredCRH2gene,fifthancestral ofthefamilyincluding history deduce theevolutionary locations acrossspecies.Thisapproachallowedusto phylogenetic analysesandcomparisonofchromosomal applied ourcombinedapproachofsequence-based ofthepeptidefamily. history evolutionary We, therefore, lineages. Thishasledtoconflictingmodelsofthe its membersandgenelossesindifferentevolutionary ratesamong difficult toresolvedueunevenevolutionary The evolution of the CRH family has been exceedingly Discussion et al. 2010 , et al.

Grone &Maruska2015b Evolution oftheCRHfamily Published byBioscientifica Ltd. In ordertodatemoreaccuratelytheCRHfamilygene Members oftheCRHpeptidefamilywereidentified Sundstrom Lagman t al.2010, et . 2013 , et al Downloaded fromBioscientifica.com at09/25/202101:32:38PM ). et al. 2011)andwassuggested Larhammar t al. 2008, et Larhammar 57 57 et al. 2011, : : 1 1 ). al.2013). et t al. 2015) et . 2015 ) et al Sundstrom Grone . et al 82 82 via freeaccess Journal of Molecular Endocrinology 2006). Afullcolourversionofthisfigureisavailableathttp://dx.doi.org/10.1530/JME-16-0051. Lepisosteiformes-Teleostei (±325MYA); andAves-Mammalian (±320MYA). Agnatha-Gnathostomes(±500MYA) wastakenfrom(Kuraku&Kuratani http://www.timetree.org/: Elasmobranchii-Euteleostomi(±488MYA); Actinopterygii-Sarcopterygii(±436MYA); Latimeria-Tetrapoda (±413MYA); respecting theproposeddivergencetimes(millionyearsago)ofvertebratelineagesrepresentedaccordingtomeandifferent studiesgivenby remains tobeconfirmedandtheyarerepresentedbydashedcirclesCRHRmemberswereobtainedfrom(Cardosoet al.2014).Thetreewasdrawn and fortheelephantsharkCRHRgenes,datawereretrievedfromourpreviousstudy(Cardosoet al.2014).TheidentityofcyclostomeCRHfamily with acrossbecausetheywerelostintheseparticularspecies,butexistotherspecieslineages.Similarly, forzebrafishcrhr1andcrhr2 tetraploidization areshownaswhitecircles.Theucn2geneinzebrafishandtheCRH2geneschickenhumanrepresentedacolorcircles (Putnam et al.2008).Thetetraploidizationeventsaremarkedas1R,2R,and3R(teleostspecific).Genesthatweremostlikelylostsoonaftereac h regions thatcontainedtheancestralCRH1/CRH2/UCN1gene,UCN2/UCN3andCRHRgeneareshownaccordingto Proposed evolutionaryduplicationschemeforthevertebrateCRHfamilymembersandtheirreceptors.Theputativeancestralvertebrategenome Figure 4 species, see(Dreborg involve someadditionalrearrangementsinofthe 3becausethey gene familiesarenotincludedinFig. opsin paralogon, respectively. The opioid and opsin have beencalledbyustheopioidparalogonand Hence, thetwoparalogonsharboringthesegenefamilies simultaneous (Hwang gene familiestoshowthat their geneduplicationswere not performphylogenetic analyses oftheneighboring proposing originin2Rhad not identifiedCRH2anddid this (Grone&Maruska2015 b not provide syntenyandparalogon information to show proposed thatthetwosubfamiliesarosein2R,butdid ). A recently published report on CRH2 paralogon (Fig. 3 and theUCN2/UCN3subfamilyispresentinopsin CRH2/UCN1 subfamily is located in the opioid paralogon Lagman http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0051 Research . 2013, et al Larhammar . 2008, et al et al. 2013). j ). Thepreviousreport

. 2015).TheCRH1/ et al c Sundstrom

cardoso Printed inGreatBritain andothers . 2010, et al topologies are highly similar for all of these gene families reasonably closeproximityto oneanother. box, anditcanbeseenthat manyofthegenesarestillin regions. Thepositionsofthe genesareshownbeloweach as betweenspeciesforthecorrespondingchromosomal between the four chromosomes in each quartet, as well surprising thatsomedifferencesingeneorderhavearisen (Ocampo Dazaet al. 2012, intrachromosomal rearrangementshappenfrequently ofsynteny.exposing theconservation Itiswellknownthat the paralogonrelationship,andbetweenspecies,thus the chromosomeswithineachspecies,thusdisplaying been adjustedtohighlightthesimilaritiesbothbetween . Theorderofthegenesonchromosomeshas Fig. 4 3andSupplementary subfamilies areshowninFig.

Evolution oftheCRHfamily Published byBioscientifica Ltd. It should be emphasized that the gene family The chromosomalregionsforthetwoCRHgene Downloaded fromBioscientifica.com at09/25/202101:32:38PM Schmid et al. 2015),soitisnot 57 : 1 83 via freeaccess Journal of Molecular Endocrinology further details(Dreborg by chromosomedoubling;seeourpreviousarticlesfor to addfurthersupportconcomitantduplications phylogenies havebeenanalyzed in detailbyusandfound are presentintheseparalogonsandtheirsequence-based 2R tetraploidizations. Several additional gene families complete quartets,fourtriplets,andtwopairs,afterthe small subset of the neighboring gene families, with four 3arejusta between thechromosomesshowninFig. should also be noted that the paralogon relationships byduplicationinthe2Revents.It history evolutionary 3),therebysupportingshared Fig. (Supplementary described inmammals,and only recentlyaputativegene in severalvertebrateclasses. Thispeptidewasinitially absence maybeduetoincomplete genomeassemblies. explored, anditmustalsobekeptinmindthatgene consequences of these gene losses have not yet been chicken, The X. tropicalis and the anole lizardand also from zebrafish. anole lizard, chicken, and platypus; UCN1 geneisabsentfromthegenomesofX.tropicalis, CRH2 hasbeenaffectedbylineage-specificevents.The the genomesanalyzed,evolutionofUCN1, While theCRH1andUCN3geneshavepersistedinallof Lineage-specific differences inCRHfamilyevolution some lineages. and this may in turn lead to clueshow it could be lost in hopefully makeitpossibletoaddress its functional role, persistence ofCRH2inatleastfourvertebratelineageswill in ray-finnedfishesandplacentalmammals.The CRH2 hasbeenlostindependentlyatleasttwice,namely birds with the exception of chicken. This suggests that and opossumaswellintheanolelizardalso found thattheCRH2geneisalsopresentinplatypus al.2011, et acid changesandchromosomal organization (Amores genomes evolveslowlyintermsofbothrateamino no coincidence,becauseithasbeenfoundthatthese shark, thespottedgar, andthecoelacanthisprobably three vertebratelineagesrepresentedbytheelephant The presence of the newly discovered CRH2, thefifthancientmemberofpeptidefamily Larhammar et al.2015,Sundstrom2010). DOI: 10.1530/JME-16-0051 http://jme.endocrinology-journals.org Research Research In thisstudy, orthologs oftheUCN2wereidentified CRH2 geneisabsentfromplacentalmammals, X. tropicalis, and teleost fishes. Thephysiological Venkatesh . 2008, et al t al.2014).Interestingly, we et j

UCN2 is absent from cardoso Lagman CRH2 gene in the Printed inGreatBritain andothers . 2013, et al UCN2, and

JME-16-0051. This islinkedtotheonlineversionof thepaperathttp://dx.doi.org/10.1530/ Supplementary data power fortheancientvertebrategenecomplexity. doublings haveonceagainprovidedgreatexplanatory lineages. The vertebrate genome and evolutionary ratesbetweenthepeptide familymembers evolutionary have resultedindistinctgenerepertoiresandvariable tetraploidization, aswelllineage-specificgenelosses, Additional genegainsinteleostfishesthroughtheirthird its functionalroleswillbeinterestingtoexplore. placental mammals,chicken,andteleostfish.Therefore, nonplacental mammals),althoughithasbeenlostin fish (spottedgar),andseveralamniotes(birds,lizard, cartilaginous fish(elephantshark),coelacanth,ray-finned still existsinfourseparatevertebratelineages,namely 4).ThefifthCRH2gene 500 millionyearsago(Fig. displayed impressivecomplexityalreadyapproximately that resultedfromthesameevents,CRH/UCNsystem progenitor. Thus,togetherwiththetwoCRHreceptors distinct peptidegenesthatwerepresentinthevertebrate as aresultofthetwoancientgenomedoublings CRH familygenesexistedattheoriginofvertebrates Analysis ofvertebrategenomessuggeststhatfiveancestral Conclusions and weusethesamenameasthemforthisgene:crha. was drawnindependentlybyGroneandMaruska(2015a), gene isindeedaduplicateofCRH1.Thesameconclusion ded from our synteny and paralogon analyses that this several additionalteleostsincludingzebrafishandconclu duplicated inthisevent.We hadidentifiedthisgenein Tcn arose before the teleost tetraploidization, but was not phylogenetic analysesledtheauthorstoproposethat The genewasreportedtobemissinginzebrafish. and was named teleocortin (Tcn) (Hosono peptide familywasreportedinafewspeciesofteleostfish elephant sharkgenomeassembly. gene asbeingpotentiallyfunctionalinthemostrecent & deLannoy2013).However, wecouldidentifytheUCN2 codon in the coding sequence (Nock described asaresultofthepresenceprematurestop elephant shark,aputativenonfunctionalUCN2genewas (Hwang was predictedinchickenandseveralteleostgenomes

Evolution oftheCRHfamily Published byBioscientifica Ltd. After completionofthisstudy, amemberoftheCRH . 2013, et al Lovejoy &deLannoy2013).Inthe Downloaded fromBioscientifica.com at09/25/202101:32:38PM . 2011, et al 57 57 : : 1 1 t al. 2015). et Lovejoy 84 84 via freeaccess

Journal of Molecular Endocrinology Grone BP & Maruska KP 2015 b Asecondcorticotropin-releasing &Maruska KP Grone BP 2015aDivergentevolutionoftwo &Maruska KP Grone BP 2013Corticotropin-releasing factor-relatedpeptides, &Lowry CA Fox JH 1985Confidencelimitsonphylogenies:anapproach using Felsenstein J 1990Physiologicalandbehavioralresponses to &Berridge CW Dunn AJ 2008Evolutionof &Larhammar D Larsson TA Sundstrom G, Dreborg S, Hollis JH, Heard AD, Stevens SL, Heldwein KA, Kesterson RA, Coste SC, Dellu F, Koob GF,Contarino A, Smith GW, Lee KF, &Gold LH Vale WW Cardoso JC, Felix RC, Bergqvist CA & Larhammar D 2014Newinsights &Larhammar D Bergqvist CA Felix RC, Cardoso JC, Amores A, Catchen J, Ferrara A, Fontenot Q & Postlethwait JH 2011 &Postlethwait JH Fontenot Q Ferrara A, Catchen J, Amores A, 2005ProtTest: &Posada D Abascal F, selectionofbest-fit Zardoya R References manuscript. performed the analysis and discussed the results. J C R C and D L wrote the J CRC,AB,andFcollectedthedata.F, andDL Author contributionstatement grant supportfromTheSwedishResearchCouncil. Multi/04326/2013) andRCFbyFCTSFRH/BPD/89811/2012.DLhasreceived received grantsupportfromanauxiliaryresearchcontract(CCMAR/UID/ Science and Technology through project UID/Multi/04326/2013. J C R C has This studyreceivedPortuguesenationalfundsfromFCT–Foundationfor Funding perceived asprejudicingtheimpartialityofresearchreported. The authorsdeclarethatthereisnoconflictofinterestcouldbe Declaration ofinterest http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0051 Research 1143. gar fish,thespotted expressed inthehindbrainofabasal neopterygian acrossvertebrateclassesand hormone gene(CRH2)isconserved Frontiers inNeuroscience corticotropin-releasing hormone(CRH)genesinteleostfishes. Neuroscience serotonergic systems,andemotionalbehavior. Frontiers in the bootstrap.Evolution (doi:10.1016/0165-0173(90)90012-D) anxiety orstressresponses?BrainResearch Reviews corticotropin-releasing factoradministration:isCRFamediatorof pnas.0805590105) vertebrate opioidreceptors.PNAS 24 403–409.(doi:10.1038/74255) lacking corticotropin-releasinghormonereceptor-2.Nature Genetics adaptations tostressandimpairedcardiovascularfunctioninmice 2000Abnormal et al. Hohimer AR, Pantely GA, Hill JK, Murray SE, 2698–2702. intake inducedbyCRFinCRFR1-deficientmice.Endocrinology 2000 Dissociationoflocomotoractivationandsuppressionfood (doi:10.1016/j.ygcen.2014.09.004) metazoans. hormone) andinvertebrateDH44(diuretichormone44)receptorsin into theevolutionofvertebrateCRH(corticotropin-releasing genetics.111.127324) duplication. sequencing: spottedgar, anoutgroupfortheteleostgenome Genome evolutionandmeioticmapsbymassivelyparallelDNA (doi:10.1093/bioinformatics/bti263) models ofproteinevolution.Bioinformatics (Lepisosteus oculatus). Journal ofComparativeNeurology (Lepisosteus oculatus).Journal (doi:10.1002/cne.23729) General andComparativeEndocrinology (doi:10.1210/endo.141.7.7653) 7 169.(doi:10.3389/fnins.2013.00169) Genetics 188 799–808.(doi:10.1534/ 9 365.(doi:10.3389/fnins.2015.00365) 39 783–791.(doi:10.2307/2408678) 105 15487–15492.(doi:10.1073/ j

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