CNTNAP2 Is a Direct Foxp2 Target in Vitro and in Vivo In
This articleisprotected bycopyright.Allrights reserved. Accepted Article
downregulation of downregulation Department for Animal Behavior, Freie Universität Berlin, Berlin Germany Germany Berlin Berlin, Universität Freie Behavior, Animal for Department Iris Adam Iris speech impairmentspeech of and learning song renders Italso densities. spine zebrastriatal finch the song X, anucleus of Area in FoxP2 of downregulation Experimental research. language and speech for model neurobiological as a serve tractable Songbirds disorder. language and speech asevere and cause basal ganglia, of Mutations Abstract 1 the avian the avian also downregulated of regulation down natural addition, In in humans. gene target a FOXP2 maintenance. maintenance. FoxP2 genetarget in songbirds, likely affectingsyna article as doi: 10.1111/gbb.12390 differencesbetween version lead to this may been through the copyediting, typesetting, pagination andproofreading process, which This article hasbeen accepted for publication andundergone full peer review but has not Corresponding author author Corresponding CNTNAP2 1 CNTNAP2 , Ezequiel Mendoza, Ursula Kobalz, Sandra Wohlgemuth, Constance Scharff Scharff Constance Wohlgemuth, Sandra Kobalz, Ursula Mendoza, , Ezequiel FOXP2
isadirect FoxP2 promoter promoter are associated with altered brain struct brain altered with associated are CNTNAP2 patientscarrying in Area X using lentiviral vectors leads to reduced expression of expression toreduced leads vectors lentiviral using X Area in complex regulation by age and activity andactivity byage regulation complex expression. Furthermore, we report that FoxP2 binds to and activates activates and to binds FoxP2 that we report Furthermore, expression. in vitro FoxP2 . Taken together thesedataestablish. Taken together FOXP2 target mutations. Here we show that experimental experimental that show we Here mutations.
control circuitry, affects synaptic transmission and and transmission synaptic affects circuitry, control production inaccurate and imprecise, similar to to similar the imprecise, and inaccurate production in vitro
and the Versionof Record. Please cite this ptic function relevant for song learning and song and learning song for relevant function ptic and ure, including the striatal part the of part ure, including striatal the in vivo FoxP2 in zebra finches: finches: inzebra CNTNAP2 byage by or singing as a direct asa CNTNAP2 , This articleisprotected bycopyright.Allrights reserved. Accepted Article and a lower spine density in cortical layer 5b ne 5b layer cortical in density spine a lower and (Anderson leads decreaseindendritic to a cultures neuron Penagarikano potassium channels at the juxtaparanodes of myelinated axons (Girault axons myelinated of juxtaparanodes at the channels potassium gated voltage of clustering forthe is relevant CNTNAP2 understood. well yet is not pathologies these to relates function CNTNAP2 How CDFE). and Tourette autism, (e.g. language affect (Rodenas-Cuadrado (ADHD) disorder hyperactivity deficit attention epilepsy (CDFE), language specific focal dysplasia cortical schizophrenia, autism, as such disorders human in diverse implicated is and the growth and maintenance of dendritic spines (Anderson spines of dendritic maintenance and growth the and 2008, Poliak ( al. (Mukamel humans and mice in screens Foxp2 end,over800direct candidate Towards this language. human of underpinnings themolecular addressing start to point entry an provides and DVD and mutations monogenic,autosomal dominant way. This make speechand language disorder (Lai FOXP2 cause factor the transcription of Mutations Introduction less (Alcock less (Watkins tasks repetition word in poorly particularly perform and language, and speech of aspects perceptive and productive in deficits CNTNAP2 , 2008, Vernes ), a theneurexinpro ), member of etal. et al. et et al. et al. et , 2012). Loss of function in mice ( inmice 2012). offunction , Loss etal. , 2000), ornot affected (Watkins , 1999), in but important , 1999), also plays roles , 2011, Rodenas-Cuadrado , 2011, , 2011, Vernes impairment Gi (SLI), impairment epilepsy, et al. et et al. et et al. , 2001). Patients with with Patients 2001). , et al. et , 2007). Among them is contactin-associated 2is protein-like Amongthem 2007). , tein family (Suedhof, 2008, Vernes 2008, (Suedhof, tein family , 2011, Roll , 2011, et al. , 2002). Interestingly,, 2002). learning of other motor tasks is urons compared to litter-mate controls (Gdalyahu controls litter-mate to compared urons branching, spine head width and synaptic strength strength synaptic and width head spine branching, Cntnap2-/- etal. s it possible to study the causality between between causality the study to possible it s Developmental Verbal Dyspraxia (DVD), a severe a Dyspraxia (DVD), Verbal Developmental target genes have been identified in large scale havetarget genesscale beenidentified inlarge , 2014). Knockdown of of Knockdown 2014). , , 2002). The disorder is inherited in a in inherited is disorder The 2002). , et al. et neuronal migration, network formation, formation, network migration, neuronal ) results in fewer striatal interneurons interneurons fewer striatal in ) results , 2010, Spiteri FOXP2 et al. lles de la Tourette syndrome and la Tourette syndrome lles de et al. et mutations have profound mutations , 2012, Gdalyahu et al. , 2014), some of which Cntnap2 et al. et , 2003, , 2003, Horresh et al. , 2007,, Vernes , 2008). 2008). , in primary primary in et al. et CNTNAP2 , 2015, , 2015, et al. et FOXP2 et et ,
This articleisprotected bycopyright.Allrights reserved. Accepted Article in Area X (Haesler X Area in Studying 2015). al., et (Gdalyahu stabilized be to fail spines generated newly because not are eliminated, al. sapiens The FoxP2 nomenclature follows the proposed in convention Kaestner Nomenclature Methods a correlated a correlated correlated to the and regulate that canbind to FoxP2 Expression levels of effectof observed that the the possibility raising spine density in mice (Gdalyahu mice in density spine reasons: (1) 2010). We hypothesized that (Haesler song of production and learning the impairs and density spine decreased to leads finches X of zebra Area nucleus et al. (Adam vocalizations learned for expression FoxP2 of striatal importance on the light shed have Songbirds maintenance. song and learning forsong relevant are functions above the of which toilluminate promise isinteraction. a direct theresult of , 2015, Penagarikano , 2013, Wohlgemuth , 2013, et al. et , Foxp2in CNTNAP2 , 2016, Haesler CNTNAP2 CNTNAP2 FoxP2 et al. et Mus musculus Mus as a potential target of FoxP2 in pos targetof FoxP2 a potential as expressionadult males.A inAreaXofand juvenile CNTNAP2 CNTNAP2 is a target of FOXP2 in humans (Vernes humans in FOXP2 of a is target downregulation, suggesting that suggesting downregulation, , 2004, , 2004, Panaitof et al. et et al. et et al. et et al. et , 2011). The decrease in spine in densit spine decrease The 2011). , CNTNAP2 and , 2014).Experimental alteration FoxP2of levels in the striatal song et al. et and FoxP2 in all other species, including zebra finches ( finches zebra including species, other in all FoxP2 and , 2007, Heston & White, 2015, Teramitsu & White, 2006, Thompson 2006, & White, Teramitsu 2015, Heston &White, 2007, , , 2007, Heston & White, 2015, Murugan 2015, &White, Heston 2007, , FoxP2 , 2015) as does FoxP2 in zebra finches (Schulz finches zebra in FoxP2 as does 2015) , et al. et is adirect FoxP2 target gene insongbirds for the following CNTNAP2 in adult zebra finches are both higher in the striatum than than striatum in the higher both are finches zebra adult in , 2010, Thompson , FoxP2 onspinesismediated via CNTNAP2.(3) promoter the striatal regulation of regulation thestriatal tembryonic and adult songbirds holdstheadult tembryonic andsongbirds in vitro in et al. et etal. y occurs because existing spines spines existing because occurs y , 2008).(2) CNTNAP2 affects and that that and , 2013). Here we demonstrate demonstrate we Here 2013). , FoxP2 et al. et al. CNTNAP2 knockdown can lead to lead can knockdown , 2000: FOXP2 in , 2000: FOXP2 , 2013, Schulz 2013, , CNTNAP2 et al. expression is , 2010), , 2010), Taeniopygia Taeniopygia by FoxP2 FoxP2 by etal. Homo Homo ,
This articleisprotected bycopyright.Allrights reserved. Accepted Article refer to the protein. after they sang more than 500 motifs within 2h af 2h within 500motifs after than theysangmore overdosed were and song sing undirected to were allowed group singing the in Birds later. hours two singisoflurane immedi with were overdosed either not did that Birds activity. vocal morning monitor to overnight boxes recording attenuated sound At the beginning of the experiments, libitum water food provided and with light:dark-cycle a 12h:12h under aviaries large freeflight in housed in usedth (TierSchG). 39finchesmale were zebra experiments wereperformed All Subjects guttata 200 (Haesler construct control anon-silencing with hemisphere vector carrying one of twoFoxP2-knockdown constructs(shFoxP2-f or shFoxP2-h),in the other Area into injected finches were X (Adam previously Injections oflentiviral vectors mediating a Injection oflentiviralvectors acute slices. Subsequently, microbiopsies ofArea X were taken and stored individuallyat -80 into cut and extracted was quickly brain the overdosed, were day 35birds At siblings. and parents biological oftheir inthe presence upnormally grow to allowed and cages home their to transferred the choice of recorded using the Sound usingtheAnalysis software Pro recorded and monitored werecontinuously Vocalizations on. went lights the after 2h within besung to
nL per site) were placed into 8 sites in each hemisphere. Injection side and order as well as the the as well orderas and side Injection hemisphere. each in sites 8 into placed were site) per nL . Birds were sexed on the day of hatching via PCR as previously described (Adam described previously as PCR via hatching of the day on weresexed Birds . ). For all genes we use the italicized letters to letters italicized usethe we genes ). Forall FoxP2 et al. et -knockdown construct were After the animalsrandomized. surgery -knockdown were , 2016,Haesler with two different lentiviral vectors, in one hemisphere with the the with twodifferentinhemispherewith one lentiviralvectors, in accordance with the in accordance guidelin all birds (except those thatunder those birds (except all et al. , 2007). In brief, at post hatch day 23 (day 23) male zebra at hatch post male day Inbrief, zebra 23 23) , (day 2007).
(Tchernichovski FoxP2 ter theirfirst motif of th is study. Prior to the experiments, animals were animals were to theexperiments, Prior is study. atelyor after lights the in wentmorning on the refer to the gene or cDNA and roman type to to type cDNAandroman the gene or refer to -knockdown were performed as described described as wereperformed -knockdown et al. et et al. et , 2007)., Injections (approximately , 2000). went surgery) were isolated in in isolated were surgery) went es of the governmentallaw es of e day.e The first motif had et al.
°C. °C. , 2014). ad This articleisprotected bycopyright.Allrights reserved. Accepted Article brains were frozen at frozen were brains asRelative Luciferase Activity(Luciferase RLU/RenillaRLU). calculated was activity Luciferase wells. other all from subtracted was vectors expressing Renilla or Luciferase with transfected not wells from background Mean (Promega). Kit Luciferase Glo Dual the using GENios) (Tecan, reader in a plate measured were activity Renilla and Luciferase transfection, (pH 8), 0.2 protein was preincubated with 0.5 with preincubated protein was competition assay200 competition 200 and transfected with 30 and transfectedwith and lysates were affinity purified via the HIS-V5 the via purified affinity were lysates and wereHEK293T transfectedcells wi Electrophoretic assay mobilityshift (EMSA) Microbiopsies were taken as previously described (Adam described previously as taken were Microbiopsies with in conjunction wereused data expression study a (Adam for previous generated were samples All RNA Microbiopsies, RNA-extraction, cDNAsynthesis 2x10 at plate a 96well in seeded was 2011) & Arnold, (Itoh G266 line cell finch The zebra Luciferase assays protein-DNA complexes was carried out on a4 on out carried was complexes protein-DNA and either 125 inspected under formicroscope the bold letters) were incubated in binding buffer (20 buffer in binding wereincubated letters) bold TATTAT 5’- sequence (oligo probe CNTNAP2 labelled individually at at individually
µm sections on a cryostat. Microbiopsies (1 Microbiopsies a on cryostat. sections µm TATTTATTTTT
% Tween-20,10
ng of pcDNA3.1-FoxP2-Flag or empty vector (Mendoza emptyvector or pcDNA3.1-FoxP2-Flag ng of − 80
°C. Remaining sections were stored in 4 stored were sections Remaining °C. GTACTCTACATTCCTTGT −
ng of unlabeled probe were added to the reaction. For the supershift assay, assay, supershift Forthe thereaction. to added were probe of unlabeled ng
80 ng pGL4.13-SV40-Luc or pGL4.13-CNTNAP2-Luc, 30 pGL4.13-CNTNAP2-Luc, or pGL4.13-SV40-Luc ng
°C in Tissue-Tek O.C.T. compound (Sakura Finetek) and cut sagittaly into into sagittaly and cut Finetek) (Sakura compound °C inO.C.T. Tissue-Tek
mM (NH th pcDNA4-FoxP2-V5-HISB (Haesler pcDNA4-FoxP2-V5-HISB th
µg anti-V5 antibody µg anti-V5 prior tothe binding reaction. Separation of proper targeting of the biopsy. targetingof proper the 4 ) 2 SO 4 , 1 TATTTGA
another target,the reelin VLDLR. receptor % polyacrylamide Tris/glycine/EDTA gel. gel. Tris/glycine/EDTA % polyacrylamide tag. One µg purified FoxP2 protein and 0.8 and protein One µgpurified FoxP2 tag.
mM DTT) for 15’mM DTT) atroomtemperature.Forthe
mm diameter) of Area of Area diameter) mm stored and excised were X
mM Hepes KOH (pH 7.6), 30 TACT-3’, FoxP2 binding sites are indicated by by indicated are sites binding FoxP2 TACT-3’, et al. et
% (w/v) paraformaldehyde solution and and solution paraformaldehyde % (w/v) et al. et , 2016, Olias 2016, , etal. et al. , 2016), in which the , 2015)., 48hafter
ng pGL4.75-CMV-Renilla , 2007) or empty vector vector empty 2007) or , et al.
mM KCl, 1 , 2014). Briefly, 4
cells/well cells/well
mM EDTA EDTA mM
ng ofDIG FoxP2
This articleisprotected bycopyright.Allrights reserved. Accepted Article cycles of 30’’cycles95 of at MX3005P system (Agilent) using the foll the using (Agilent) system MX3005P (5’-GAGGGCAAGGTCAGTGTCCA-3’ / 5’-GAATCGAACTTCATGCCACTGC-3’). were Reactions run on a (5’-AGAACGGCATCAAGGTGAAC-3’ /5’-TGCTCAGGTAGTGGTTGTCG-3’)(Adam (5’-GCAGCATGTTGGCATCACAG-3’ / 5’-TGCTTTGCTCCCTTGCTCAG-3’) (Haesler (5’-CCTGGCTGTGAAAGCGTTTG-3’5’-ATTTGCACCCGACACTGAGC-3’) / (Haesler for amplification specificity. specificity. for amplification synthesis was carried out using random hexamer primers and 180 and primers hexamer random using out carried was synthesis extrac RNAwas total cases all In were pooled. of one hemisphere microbiopsies targeted properly all animals, For other biopsies. individual from Total RNA properlytargeted from randomly. chosen was side The sampled. was bird per hemisphere one only birds, unmanipulated In thecaseArea animals, virus-injected of fr X qRT-PCR reactions were run in duplicates in a total reaction volume 20 of volume ina reaction total induplicates run were qRT-PCR reactions qRT-PCR were added to 15 to were added water (10-fold for pooled, 5-fold for individual microbiopsies). microbiopsies). for forindividual 5-fold water pooled, (10-fold free with nuclease werediluted All cDNAs for DNA contamination. genomic to control included were reactions free Reverse-transcriptase respectively. samples, microbiopsy individual pooled or 10 prior to all measurements using 10-fold dilution seri dilution 10-fold using to allmeasurements prior included,if samples were run on differentplates. The efficiency ofeach primer pair was checked always was (IRC) calibrator inter-run An respectively. ofreagents, contamination and contamination no as well as free samples Reverse-transcriptase all primer pairs ranged within 2 within ranged pairs primer all
pmol of each primer (18 primer each of pmol
µL reaction mix containing10
°C, 30’’at 65
pmolin the case of
°C (64 °C microbiopsies ofbirdswith a
°C for
±10 owing temperature program: 10‘ at95 program: temperature owing
%. We used the following primer pairs: pairs: primer following used the %. We FoxP2
FoxP2 om both hemispheres was sampled, in the case of caseof inthe sampled, was hemispheres both om µL 2xKAPA SYBR FAST Universal QPCR Mix (Peqlab), ted using ted cDNA the RNAXSkit (Macherey-Nagel). template totestcontrols wereincluded for DNA- and and 60 es over 6 ordersmagn over es of ) and ROX (50 ROX and )
°C for HMBS FoxP2
nM, Peqlab)asa reference dye.
ng or 40 ng or ) and a melting curve to melting curve to ) anda check knockdownwas extracted
µL. Five cDNA µLof µL. diluted et al. et itude. The efficiency of
et al. et ng total RNA forng totalthe RNA etal.
°C followed by 40 , 2016), , 2016), , 2007), 2007), , , 2007), , 2007), CNTNAP2 HMBS FoxP2 GFP
This articleisprotected bycopyright.Allrights reserved. Accepted Article juvenile Area Area set data (Hilliard juvenile microarray a published using versa, X and vice the to (Smedley Biomart Yates (ENS87, annotation Ensembl to the current according homolog finch a zebra with factors transcription of only matches and retained then table (Cartharius MatInspector and expression values that are stro expression values are that mRNA with factors transcription weremoved step last a In data. transcriptome unpublished own GOIs: 2016, Haesler as it is ofstable the most all tested poten expression thegene for of interest (GOI) ( The meanC binding sites by means of three databases - Jaspar (Mathelier -Jaspar databases of three bymeans sites binding taeGut WashU chr2:31,216,312-31,217,544, location: We utilized the Search transcriptionfactorbindingsitesinthe for GFP in both hemispheres. hemispheres. both in GFP Individual biopsies ofvirus-inje biopsies Individual hemisphere. and animal per averaged were values expression Relative used to to measure theexpressionused of successfulinfection prior torunningall other assays. OnlycDNA from CNTNAP2 t for each sample was derived from the run run the from derived was sample foreach et al. CNTNAP2 promoter by keeping only transcription fa transcription only keeping by promoter et al. , 2007). We used the following formula to to formula following Weused the , 2007). , 2015). We further filtered the list of transcription factors potentially binding binding potentially factors list of transcription the filtered further We , 2015). promoter sequence that we used in the Luciferase experiment (genomic (genomic experiment inthe weused Luciferase that sequence promoter et al. ngly correlated with those of . . . cted animals were screened for for screened cted animalswere , 2005) - using the default settings. We merged all results into one FoxP2 , FoxP2 CNTNAP2 tial reference genes for ourexperiments (Adam = or ( et al. et ( CNTNAP2 and and , 3.2.4/taeGut2) to predict transcription factor factor transcription topredict 3.2.4/taeGut2) , HMBS CNTNAP2 , 2016).Homologs were identified using data and used to calculate relative gene gene relative calculate to used and data ctors that are expressed in adult, but not not but adult, in expressed thatare ctors , , FoxP2 , , et al. et ). We used ). We calculate the relative expression of our of expression calculate relative our the . 84 (orange Module in (orangeModule (Hilliard ) , 2016), Patch (Matys Patch , 2016), GFP
% of all injected birds expressed expressed birds injected all of % )
promoter expressionafor as marker GFP HMBS -positive biopsies were were biopsies -positive et al. as a reference gene, asareference , 2012),and our et al. , 2006) et al. et et al. et , , This articleisprotected bycopyright.Allrights reserved. Accepted Article changed its transcriptional activity. We performed these experiments on a zebra finch cell line line cell finch a zebra on experiments these performed We activity. its transcriptional changed the to FoxP2 of binding if test to assays reporter luciferase used further We a supershift of the band (“ss”), confirming that FoxP2-protein caused the initial shift. to led FoxP2-V5 against an antibody with Pre-incubation (“competitor”). probe unlabeled of addition regulatory regions of the zebra finch regulatory regions ofthe zebra binds to these sites, we conducted electrophoretic mobility shift assays (EMSA). We designed a 46 a designed We (EMSA). assays shift mobility electrophoretic conducted we sites, tothese binds 5’-UTR-reg in the in particular sites, binding FoxP2 probe containing three binding sites approximately 350 approximately sites binding three containing probe labeled probe shiftin anupwards ( resulted theband of the to protein FoxP2 Adding taeGut3.2.4/taeGut2). WashU chr2:31,216,574-31,216,619, location: (Wickham, 2009). using after knockdown) FoxP2 expression) using using CNTNAP2) and FoxP2 using were conducted assays) tests (Luciferase an data the using wereperformed tests All statistical analysis Statistical of regulators potential finding in interested wewere because 2012)), investigate investigate thisin zebrafinches, screened for we sites FoxP2 binding (Nelson regulate can FoxP2 that and regions regulatory for prerequisite A enhancing directly is FoxP2 Results of downregulation FoxP2’s counteract or ofFoxP2 independently CNTNAP2 lm() wilcox.test() to be a target FoxP2 tobe ishasgenethatit binding FoxP2 sitesinits wilcox.test() CNTNAP2 and Wilcoxon signed rank tests (reduction of FoxP2 and CNTNAP2 and CNTNAP2 FoxP2 of (reduction rank tests signed and Wilcoxon CNTNAP2 expression . Plots were generated using the ggplot2 package usingtheggplot2 package . Plots were generated gene (ENSTGUG00000001794). We identified several several identified We (ENSTGUG00000001794). gene CNTNAP2 ,regressions (dependency of CNTNAP2 expression on on expression ofCNTNAP2 (dependency ,regressions t.test() ion of ion gene. the Totest whether FoxP2protein the in vitro in alysis software R (R Core Team, 2013): Paired 2013): t- R Team, (R Core software alysis
bp upstream of the start-codon (genomic (genomic thestart-codon of upstream bp Figure 1a expression bybinding to thesemotifs. To , Mann Whitney U tests (age difference difference (age U tests Whitney Mann ,
, “ps”), which was abolished by the the by abolished was which “ps”), , CNTNAP2 CNTNAP2 . CNTNAP2 et al. et , 2013) in the inthe 2013) , that acteither that
promoter promoter
bp This articleisprotected bycopyright.Allrights reserved. Accepted Article However, However, CNTNAP2 ( p=0.002737) expression, which was significantl cells (Adam SV40 protein inG266 expression cells reduced the of the tr entire the also but finches, zebra from were FoxP2-protein and promoter the only not that ensured lines, cell human used typically the of instead line cell theG266 Choosing 2011). & Arnold, (Itoh bird male a of tissue from tumor derived (G266) test, U=120, p=0.0009774) ( p=0.0009774) test, U=120, juvenile in than n=9) days, 297 mean days, 968 finding that previous our confirmed on the day of sacrifice. After takingmicrobiopsies from Area Xwe performed qRT-PCR. The results sing not did males that sure wemade set, data onthis song undirected of influence the To exclude of hypothesis,levels bo wemeasuredthe expression we would expect tosee positively correlated changesin al. Thompson FoxP2 expression inAreaXofad islower CNTNAP2 negative correlation between the between expressionnegative correlation the of levels directly regulate regulate directly ( p=0.0078) df=5, 4.2861, in presencethe of butnot inthepresence the of FoxP2, vector empty control (paired t-test, n=6, t=- of the , 2010, Teramitsu & White, 2006). If FoxP2 directly regulated regulated directly FoxP2 If 2006). & White, Teramitsu 2010, , promoter (paired t-test, n=6, t=3.1417, df=5, t-test, n=6, t=3.1417, ( promoter (paired p=0.0256) CNTNAP2 expression in AreaX of malezebra finches. FoxP2 and etal. et al. et Figure 2b FoxP2 promoter including the 5’-UTR we found a significant enhancement of transcription transcription of enhancement significant a found we 5’-UTR the including promoter and , 2013) and is downregulated by undirected singing in both age groups (Teramitsu in age groups both singing by undirected is downregulated and , 2013) CNTNAP2 , 2016)., When we tested the FoxP2-dependent transcription from1,591 a CNTNAP2 expression are positivelycorrelated inof Area X juvenilemales. ). This direction of regulation suggested that FoxP2 may be repressing berepressing may FoxP2 that suggested ofregulation direction This ). Figure 1b Figure expression in zebra finches. finches. zebra in expression Figure 2a levels were strongly positively correlated in juvenile individuals individuals juvenile in correlated positively strongly were levels y higher in adults compared to juveniles (Mann Whitney U test, U test, Whitney (Mann tojuveniles in compared adults higher y , CNTNAP2). We thus conclude th conclude thus We CNTNAP2). , FoxP2 FoxP2 ) (Haesler ult males than in juvenile males (Haesler males in juvenile than males ult levels in Area X are lower in adult birds (>120 days, 123- (>120 days, in adult birds Xarelower in Area levels anscriptional machinery. Overexpression of FoxP2 of FoxP2 machinery. Overexpression anscriptional s (50-52 days, mean 50 days, n=15) (Mann Whitney U Whitney (Mann 50 n=15) days, mean days, s (50-52 et al. et luciferase reporter gene under the control of of the the control under gene reporter luciferase , 2004). The opposite was the case for case the The oppositewas , 2004). th genes inth genes Area X of juvenile andadult males. If werethis the case, FoxP2 CNTNAP2 and and Figure 1b Figure CNTNAP2 CNTNAP2 CNTNAP2 CNTNAP2 mRNA levels. To test our at FoxP2 has the capacityto at hasthe FoxP2 , SV40) as it does in HEK293T HEK293T in does it as SV40) , inArea X of zebra finches we expected to find expected a to we in individual birds. et al. , 2004,
bp region region bp CNTNAP2 et
This articleisprotected bycopyright.Allrights reserved. Accepted Article FoxP2 ( p=0.8021) (Linear Model,R (Linear lead toa correlated downregulation of in change induced singing the whether asked thus We learning. song of Riters 2013, & Doupe, (Brainard plasticity Thompson 2006, White, & (Teramitsu finches zebra male adult in expression FoxP2 of adownregulation to leads which song, Undirected CNTNAP2 thisbirdsR was inadult Model, (Linear not the case levels of of levels adults ( adults firstmore for details) (seemotif Methods the day of the after hours two within song undirected of motifs 500 more than sang that birds adult of group et al. (Andalman song undirected or learning song like that FoxP2 interaction ofwith the mRNA levels in adult males in comparison to juveni to incomparison males adult in levels mRNA enhance which areonlyadultexpressed in X Area al. accountinto that ex are factors transcription took transcriptionfactors affecting potential silico in ( Table S1 Table , 2012, unpublished own data), (see Methods ford , 2011, Tumer & Brainard, 2007, Woolley 2007, Tumer &Brainard, 2011, , in juvenile non-singing males with the fact that levels of of levels that fact the with males non-singing juvenile in for transcription factor binding sites on the onthe sites binding factor transcription for Figure 2d FoxP2 CNTNAP2 ). and Figure 2d expression innon-singing adults?Another, unidentified,age dependentfactormight FoxP2 2 ) =0.467, adjusted R CNTNAP2 expressionadult AreaXand inthis ). How can we reconcile the findingthat the canwe reconcile ). How expression levels are positively areexpression levels undirectedcorrelated song after expression was significantly and positively correlated with with correlated positively and significantly was expression CNTNAP2 2 =0.4261, F =0.4261, et al. CNTNAP2 CNTNAP2 and have predictedbi et al. et , 2013) is thought to asso be thoughtto is , 2013) might only occur during periods of enhanced plasticity, et al. , 2014), which bears resemblance to the juvenile state state the juvenile to resemblance bears which 2014), , (1,13) expression in adult male & Fee, 2009, Brainard & Doupe, Charlesworth 2013, &Doupe, Brainard 2009, & Fee, expression in adult non-singers. We thereforein adult non-singers. only expression pressed inadults but not in juveniles (Hilliard =11.39, p=0.004973) ( p=0.004973) =11.39, le males. To address this possibility, we searched we possibility, this address To males. le , 2014). Totest this, weadded an additional CNTNAP2 2 . Indeed, in contrast to the results in non-singing non-singing in results tothe incontrast . Indeed, =0.009593, adjustedR etails). identifiedfactors, We 76transcription could cause the significantly higher could thesignificantly cause promoter to narrow down the list of listof down the tonarrow promoter CNTNAP2 nding sites innding sites the CNTNAP2 expression is enhanced by ciated with enhanced neural neural enhanced with ciated FoxP2 expression are not linked to to linked are not expression Figure 2c birds. Wehypothesized 2 =-0.1319, F mRNA levels would would levels mRNA CNTNAP2 ). Interestingly, (1,7) FoxP2 =0.0678, =0.0678, CNTNAP2 promoter promoter
et et
This articleisprotected bycopyright.Allrights reserved. Accepted Article regulated the singing vigorously. In the same vein, an experimental knockdown of FoxP2 led to lower led to ofFoxP2 knockdown experimental an vein, same In the vigorously. singing expression were positively which was correlated, R To causally link our finding that that finding our link To causally CNTNAP2 R Model, (Linear individuals singing in expression reported for humans (Vernes humans reported for whether we study investigated In this Discussion ( p=0.1422) W=18, test, rank signed when compar hemispheres knockdown we of measured the expression ( n=7) p=0.01563, W=28, test, rank signed (Wilcoxon hemisphere control tothe compared that Afterconfirming hemisphere. 23, males atdaywhileaco hemisphere ofjuvenile of decrease 2010).We expected to (Adam previously described as vectors lentiviral using males juvenile of X Area in levels FoxP2 reduced we experimentally p=0.01916) ( p=0.01916) adjusted R adjusted CNTNAP2 correlation between theexpression of the two gene correlated to the 2 =0.618, F =0.618, expression 2 expressionisreducedafter (1,5) =0.4823, F FoxP2 Figure 3 CNTNAP2 =10.71 p=0.02215) ( p=0.02215) =10.71 FoxP2 . Lentiviral vectors mediating a mediating vectors . Lentiviral ) in vivo . (1,6)
see a downregulation of expression levels in both, the control (Linear Model, R Model, (Linear control the both, in levels expression promoter. In non-singing juvenile zebra finches finches zebra juvenile Innon-singing promoter. =6.591 p=0.0502) ( et al. et .
et al. et CNTNAP2 CNTNAP2 , 2016, Haesler Figure 4cFigure , 2008). We found that FoxP2 protein directly bound to and toand bound directly protein FoxP2 that found We 2008). , FoxP2 Figure 4b CNTNAP2 ed tothecontrol hemisphere was downregulated on the knockdown hemisphere onthe downregulated knockdown was expressionis positi in all hemispheres. in all hemispheres. ) and the knockdown group (Linear Model, R Model, (Linear group knockdown the and ) Figure 4d Figure FoxP2 ). Moreovertheexpression of isdirect a FoxP2 target gene in songbirds as CNTNAP2 et al. et 2 FoxP2 alsomales whenthey in adult thecase were =0.3098, adjusted R adjusted =0.3098, ntrol construct was injected into the contralateralthe into injected was construct ntrol knockdown s. We concluded that FoxP2 can indeed regulate regulate indeed can FoxP2 that s. We concluded ) as indicated bythe )as indicated , 2007, Murugan , 2007, knockdown were injected wereintoXof knockdown Areaone mRNA levelspositively correlated to the vely correlated with correlated vely CNTNAP2
of of the sameanimal (Wilcoxon 2 et al. et =0.565, F =0.565, levels were reduced in 4 of 7in 4 of reduced were levels CNTNAP2 significant positive positive significant CNTNAP2 , 2013, , 2013, Schulz 2 =0.6817, adjusted adjusted =0.6817, (1,6) FoxP2 and and =10.09, =10.09, waspositively FoxP2 CNTNAP2 expression, 2 =0.5686, et al. et Figure 4a
,
), ), This articleisprotected bycopyright.Allrights reserved. Accepted Article observed onstriatal spinedensity (Schulz effect previously the like songbirds, male in of FoxP2 knockdown after observed changes neuroanatomical CNTNAP2 betweenFoxP2and regulatory relationship The significance. statistical reach not did difference this though even birds, 7 our of 4 in levels (Vernes cortex human of todownregulation leads The direction of regulation differs in human neur study, our In X. Area in of upregulation a concomitant the normalized situ al. Panaitof by those and findings our between discrepancy The X. Area adult and juvenile between We found remodeling complex and members of the FoxO-subf members of and remodeling complex familyand members Ct (FoxP1 FoxP4), Nkx2.1, FoxP- other be could cofactors for such Examples of regulation. direction the influencing tissues, factors (Diamond transcription binding totheasiteenhance samegene of ordependingonthepresence repress other can (Varea neurons pyramidal finches. male zebra adult singing LMAN of zebra finches (Condro & White, 2014, Panaitof 2014, & White, (Condro finches ofzebra LMAN , 2010 might be due to the fact that fact beduetothe might 2010 , hybridizations, methods with different dynamic ranges. Additionally, Panaitof Additionally, ranges. dynamic different with methods hybridizations, et al. expression in zebra finches can be regulated by FoxP2 CNTNAP2 FoxP2 , 2008). One explanation for these tissue diff thesetissue for explanation One , 2008). CNTNAP2 CNTNAP2 expression waspositively correlated with expression levels in Area X to be lower in non-singing juveniles than in non- in than juveniles non-singing in lower be to X Area in levels expression is expressedhighestinlayers withlow expression in Area X to the expression in the surrounding striatum, sothat expressionexpression AreainX tothe the in surrounding etal. CNTNAP2 et al. et CNTNAP2 , 2015),Panaitof While age related upregulation of age relatedupregulationof While , 1990). Different cofactors exist in different cell lines and and lines cell different in exist cofactors Different 1990). , expression (Vernes expression we used qRT-PCR whereas Panaitof whereas we qRT-PCR used in the striatum couldobscure theupregulation of CNTNAP2 BP1, PIAS1, members theNuRD chromatin- of members BP1, PIAS1, et al. et al. on-like cell line, where overexpression of FOXP2 where overexpression of line, cellon-like amily, all known to interact with FoxP2 and to and with amily, allFoxP2 tointeract known , 2010). may contribute to the behavioral and and behavioral tothe contribute may , 2010 did not find did expression not , 2010 differences et al. et al. erences coulderences be factors thattranscription , 2010) and in cultured murine murine in and cultured , 2010) CNTNAP2 , 2008). Likewise, in the developing developing inthe Likewise, , 2008). FOXP2 CNTNAP2 expression and vice versa expression in zebra finches. et al. was also reported in in reported also was , 2010 performed performed , 2010 et al. et , 2010, CNTNAP2 et in
This articleisprotected bycopyright.Allrights reserved. Accepted Article modulate its ability to itsabilityto regulate (Chokas targetgenemodulate expression factors are good candidates to mediate the age-dependent upregulation of of upregulation theage-dependent mediate to candidates good are factors differencesspecific Tissue determi (Mathelierrespectively sites, binding FoxO3 and FoxP1 the to similar very are which sites, binding FoxO4 and FoxP2 (Van Boxtel genes target FoxO3 th Through enhancers. same the to bind are knownto al. Hilliard data, own (unpublished finches zebra male adult as as well juvenile AreaX of in expressed al. FoxP2 correlation of correlation of and nidopallium nidopallium and (Alarcon huma of striatum and thalamus dorsal developing (Haesler Panaitof &White,2014, in(Condro expression pattern songbird brain the transcription factors with pr an used AreaX we in expressed ones for additional search FoxP2 notthe isclearly In summary, the bind to might not this effect, shown relationshipby a absence the of adults); singing and juveniles in (e.g. expression expressed, asis inAreathe case X, ourda regulation of , 2012). Especially the FoxO-family members are are members the FoxO-family Especially 2012). , , 2004, Van Boxtel expression overlap in the striatum, Purkinje ce Purkinje striatum, the in overlap expression etal. et al. et CNTNAP2 , 2004, Teramitsu , 2004, , 2008,Teramitsu FOXP2 CNTNAP2 et al. et and and CNTNAP2 . et al. et CNTNAP2 , 2013, 2013, Zhou , is expressed but edicted sitesedicted inthe binding , 2016). , 2016). et al. et al. et et al. promoter ning thedirectionof regulation levels in human cortex only transcription factor regulating regulating factor transcription only , 2004). In areas where both where areas 2004). In , , 2004). Furthermore, in the zebra finch brain brain finch zebra in the Furthermore, 2004). , , 2013). The same kind inof kind same 2013). The , et al. et ta show that FoxP2 can positively influence positively can FoxP2 that show ta
or if it does, additional factors prevent its transcriptional itstranscriptional prevent factors additional does, it if or FoxP2 , 2008)., allFurthermore, cofactors ofthese are however, FoxP2 expression does not always have always not does expression FoxP2 however, is not. In general, In isnot. general, ns where both genes can be co-expressed strongly strongly co-expressed be can genes both where ns promising candidates because FoxO3 and FoxP1 and FoxO3 because candidates promising innon singing adults.In ll layer and in opticthe whereas LMAN tectum, is interaction FoxP1 is able to regulateis able specific FoxP1 is interaction CNTNAP2
in silico (Vernes et al. et teraction might occur between teraction mightoccur promoter. promoter. These transcription approach and identified approach and 76 are also likely since the negative negative likely sincethe also are et al. et CNTNAP2 , 2010, Estruch FoxP2 CNTNAP2 , 2008) , etal. CNTNAP2 the latter the case, FoxP2 and has a much wider wider much a has , 2010) than
is not mirrored in mirrored is not expression. To expression. CNTNAP2 CNTNAP2 etal. CNTNAP2 expression. , 2016, , 2016, Li areco- FoxP2 and and
et et et This articleisprotected bycopyright.Allrights reserved. Accepted Article et al. Taking our findings and published data together, FoxP2 does regulate regulate does FoxP2 together, data published and findings our Taking genes. receptor gene (Yang receptor repres to known factor atranscription was SP3, CNTNAP2 the to bind actually list this from candidates the whether address to needed are experiments Further Adam, I., Scharff, C. &Honarmand,(2014)M. is Whowho? Non-invasive methods to individuallysex & S. U., Wohlgemuth, Kobalz, E., Adam, I., Mendoza, References interests. financial nocompeting declare The authors byCONACYT. supported was EM Neurobiology'. 'Molecular School Research International Helmholtz This worksupported bythe was DFG (EXC25 Acknowledgements on variabil injecting not is pathway forebrain anterior mo adult the state of “default the in However, influences FoxP2 males, adult in singing undirected We propose that during periods of enhanced plasticity, such as in juvenilemales and during zebra finches, yet the relationship ismore complicated thana simple “moreis more” dependency. CNTNAP2 , 2013). Being regulated by FoxP2 and SP3 might and SP3 FoxP2 by Being regulated 2013). , and mark altricial chicks. chicks. altricial mark and singing. by and developmentally VLDLR receptor reelin promoter promoter might be overridden byother might factors, e. overridden be et al. et in vivo in , 2000).Interestingly, . The candidate gene with most binding sites insites bindingmost the with gene The candidate . J Vis Exp . 7 NeuroCure, SFB665). IA was supported by the bythe supported IA was SFB665). 7 NeuroCure, tor system”tor (Brainard &2013), Doupe, when the s theSP1mediated activation of thehuman D1A D1A ity into the motor pathway, the influence of FoxP2 of FoxP2 influence the pathway, motor the into ity CNTNAP2 is regulatedin byFoxP2 zebra finches (Murugan Scharff, C. (2016) FoxP2 directly the regulates FoxP2 C.(2016) directly Scharff, g. miRNAs or other transcription factors. factors. transcription other or miRNAs g. thus be common to thus to asubset FoxP2be common of target expression in a linear manner. manner. alinear in expression Mol Cell Neurosci Cell Mol CNTNAP2 CNTNAP2 in Area X of male Xof inArea . promoter promoter
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This articleisprotected bycopyright.Allrights reserved. Accepted Article (supershi lane). Pre D labeled used in E cells tran Figure 1: Figure enhance transfect Overexpr d M e s s f e FoxP2 binds binds FoxP2 NA probe NA probe (s incubation o incubation legends transcriptio t, ss) of thel fected with with fected d with a pG a d with ssed FoxP2 SA assays. SA assays. e e L P P p n p n p f t t abeled prob abeled cond left lan left cond 4.13 plasmi the purified the resence of F of resence o andactiv cDNA4-Fox via the rotein repr rotein CN T d a e e P e oxP2 in the s protein with protein NAP2 containing t tes the ssed lucifera ssed indicating s 2-V5-HISB o 2-V5-HISB ), which wa prom CNT o o N N p p r s r s ample led to led ample to he se transcript se ter. anti-V5 anti emptyvect successfull ecific bindin PA2 SV40 promo pro y y m m o t b i i g of FoxP2 p FoxP2 of g competed b competed on in theon ze an upwardp er. r wasaffinit ody led a ody to oter. In con (a) Prote b b r i i y n additional n additional y y t rotein shift ( shift rotein n lysate fro lysate n otein to the ra finch cell rast, FoxP2 purifiedan unlabeled unlabeled m m p p d u s u p l l probe. (b) (b) probe. ine G266 G266 ine ignificantly ignificantly robe (third robe (third HEK293T subsequen pward shift shift pward s) ofthe t t ly ly
This articleisprotected bycopyright.Allrights reserved. Accepted Article denote the 0.95 thedenote interval confidence adults. representsEach dot non-singing oneindi significantly and positi significan are levels expression (b) sacrifice. to prior sing not did that males adult and juvenile Xof of Area samples microbiopsy from qRT-PCR by measured were levels Expression fill, n=9). black days, 297 mean days, fill no 50 days, mean days, (50-52 males juvenile juveniles. of non-singing Figure 2: males. juvenile letters adult group theto1. of mean and the was set CNTNAP2 expression is strongly and positively correlated to to correlated positively and strongly is expression vely correlated with (a) FoxP2 tly higher in adults mRNA expression levels in Area X are significantly higher in higher significantly are X in Area levels expression mRNA . Gene Gene expression was normalizedto the gene reference FoxP2 Upper caseletters males,denote lowercase adult vidual; the dashed lines lines dashed the vidual; , n=15) than in adult males (>120 days, 123-968 123-968 days, (>120 in adultmales than , n=15) expression in non-singing juveniles, (d) but not in in not but (d) juveniles, in non-singing expression than in juveniles. (c) Expression of of Expression (c) in juveniles. than FoxP2 around the fittedaround line expression in Area X X inArea expression CNTNAP2 CNTNAP2 HMBS mRNA mRNA was
This articleisprotected bycopyright.Allrights reserved. c Accepted Articlea
CNTNA P2 expressio n 0.2 0.4 0.6 0.8 Fold change expression 6 0 2 4 p=0.005 juv enile 1 juv FoxP2 enile 2 *** ex 3 pressio adult 4 p=0.0009774 n FoxP2 5 d b
CNTNAP2 expression 0.0 0.5 1.0 1.5 Fold change expression 1 2 0 0.4 juv FoxP2 enile 0.8 ex ** pressio adult 1.2 p=0.8021 CNTNAP2 p=0.002737 n adult This articleisprotected bycopyright.Allrights reserved. Accepted Article Figure 3:
represents one individual;represents one dashed with correlated positively and strongly was song of undirected motifs CNTNAP2 CNTNAP2 expression in adult males (248-405 days, mean 352 days, n=8) that sang more than 500 500 than more sang that n=8) days, 352 mean days, (248-405 males adult in expression expression positi is
CNTNAP2 expression deno around thefittedline lines 0.0 0.2 0.4 0.6 vely correlated with with correlated vely p=0.019 undir F ected song o xP2 0.5 e xpression 1.0 FoxP2 expression in singing adults. adults. in singing expression te the 0.95 confidence interval te the 0.95confidence FoxP2 expression. Each dot dot Each expression. . This articleisprotected bycopyright.Allrights reserved. Accepted Article Figure 4: Knockdown Figure 4:Knockdown of males. individual denote with (c) in someanimals. was decreased (white hemisphere the control compared to fill) randomized. (a) 23) (day juvenile of X Area right and left into expression eitherashRNAdire of individual; the dashed linesar FoxP2 expression in both, the control and (d) knockdown hemisphere. Each dot represents one one represents dot Each hemisphere. knockdown (d) and thecontrol both, in expression FoxP2 expressionsignificantly was decrease FoxP2 ound the fitted line denote the interval ound 0.95 fittedconfidence linedenote the reduces reduces cted against FoxP2 or a non-silencing control shRNA were injected injected were shRNA control or anon-silencing FoxP2 against cted CNTNAP2 CNTNAP2 male zebra finches. Injection order and side were were side and order Injection finches. zebra male expression was significantly and positively correlated correlated positively and significantly was expression fill) of the same animal.(b)Expression of expression levels. d on in thein knockdown d on hemisphere (no Lentiviral vectors mediating the . Numbers CNTNAP2
This articleisprotected bycopyright.Allrights reserved. c Accepted Articlea
CNTNAP2 expression 0.5 1.0 1.5 2.0 2.5
FoxP2 expression % 100 60 80 6 p=0.016 F 0.5 p=0.022 R shControl 5 o 2 7 4 =0.618 xP2 shControl F o 1.0 xP2 e * xpression 3 1.5 1 shF o 7 5 3 6 2 4 xP2 1 2.0 2 d b
CNTNAP2 expression 0.4 0.6 0.8 1.0
CNTNAP2 expression100 % 0.25 60 80 40 6 p=0.14 CNTNAP2 p=0.0502 R shF 2 =0.482 5 shControl 4 7 o 0.50 xP2 F o xP2 e 0.75 n.s xpression . shF 1 o 1.00 xP2 2 3 4 6 7 5 1 2 3