Deletion of the Wilms’ Tumor Suppressor Gene in the Cardiac Troponin-T Lineage Reveals Novel Functions of WT1 in Heart Development

cell-09-683861 July 17, 2021 Time: 18:39 # 1 ORIGINAL RESEARCH published: 22 July 2021 doi: 10.3389/ cell.2021.683861 Edi ed by: O elia M. Ma ínez-Es ada, Uni e si y o Ba celona, Spain Re iewed by: Ka he ine Yu zey, Cincinna i Child en’s Hospi al Medical Cen e , Uni ed S a es Ma ie-José Goumans, Leiden Uni e si y Medical Cen e , Ne he lands Sil ia Ma in-Puig, Spanish Na ional Cen e o Ca dio ascula Resea ch, Spain *Co espondence: Ramón Muñoz-Chápuli [email p o ec ed] Manuel Jiménez-Na a o [email p o ec ed] † † †P esen add ess: Ri a Ca mona, Depa men o Human Ana omy, Legal Medicine and His o y o Science, Facul y o Medicine, Uni e si y o Málaga, Málaga, Spain Special y sec ion: This a icle was submi ed o Signaling, a sec ion o he jou nal F on ie s in Cell and De elopmen al Biology Recei ed: 22 Ma ch 2021 Accep ed: 18 June 2021 Published: 22 July 2021 Ci a ion: Díaz del Mo al S, Ba ena S, He nández-To es F, A ánega A, Villaescusa JM, Gómez Doblas JJ, F anco D, Jiménez-Na a o M, Muñoz-Chápuli R and Ca mona R (2021) Dele ion o he Wilms’ Tumo Supp esso Gene in he Ca diac T oponin-T Lineage Re eals No el Func ions o WT1 in Hea De elopmen . F on . Cell De . Biol. 9:683861. doi: 10.3389/ cell.2021.683861 Dele ion o he Wilms’ Tumo Supp esso Gene in he Ca diac T oponin-T Lineage Re eals No el Func ions o WT1 in Hea De elopmen Sand a Díaz del Mo al1, Sil ia Ba ena1, F ancisco He nández-To es2,3, Amelia A ánega3,4, José Manuel Villaescusa5, Juan José Gómez Doblas5, Diego F anco4, Manuel Jiménez-Na a o5*, Ramón Muñoz-Chápuli1*and Ri a Ca mona1† 1Depa men o Animal Biology, Uni e si y o Málaga, Málaga, Spain, 2Depa men o Biochemis y and Molecula Biology III and Immunology, Facul y o Medicine, Uni e si y o G anada, G anada, Spain, 3Medina Founda ion, Technology Pa k o Heal h Sciences, G anada, Spain, 4Depa men o Expe imen al Biology, Facul y o Expe imen al Sciences, Uni e si y o Jaén, Jaén, Spain, 5Hea A ea Clinical Managemen Uni , Uni e si y Hospì al Vi gen de la Vic o ia, CIBERCV En e medades Ca dio ascula es Heal h Ins i u e Ca los III, Biomedical Resea ch Ins i u e o Malaga (IBIMA), Uni e si y o Málaga, Málaga, Spain Exp ession o Wilms’ umo supp esso ansc ip ion ac o (WT1) in he emb yonic epica dium is essen ial o ca diac de elopmen , bu i s myoca dial exp ession is li le known. We ha e ound ha WT1 is exp essed a low le els in 20–25% o he emb yonic ca diomyocy es. Condi ional abla ion o WT1 using a ca diac oponin T d i e (Tnn 2C e) caused abno mal sinus enosus and a ium de elopmen , lack o pec ina e muscles, hin en icula myoca dium and, in some cases, in e en icula sep um and ca diac wall de ec s, en icula di e icula and aneu isms. Co ona y de elopmen was no mal and he e was no emb yonic le hali y, al hough su i al o adul mu an mice was educed p obably due o pe ina al mo ali y. Adul mu an mice showed elec oca diog aphic anomalies, including inc eased RR and QRS in e als, and dec eased PR in e als. RNASeq analysis iden i ied di e en ial exp ession o 137 genes in he E13.5 mu an hea as compa ed o con ols. GO unc ional en ichmen analysis sugges ed ha bo h calcium ion egula ion and modula ion o po assium channels a e deeply al e ed in he mu an myoca dium. In summa y, oge he wi h i s essen ial unc ion in he emb yonic epica dium, myoca dial WT1 exp ession is also equi ed o no mal ca diac de elopmen . Keywo ds: Wilms’ umo supp esso gene, ca diomyocy es, ca diac de elopmen , calcium homeos asis, po assium channels INTRODUCTION The Wilms umo supp esso gene (W 1) encodes a C2H2- ype zinc- inge ansc ip ion ac o ha appea s in mammals unde di e en iso o ms, pa icipa ing in ansc ip ional egula ion, RNA me abolism and p o ein-p o ein in e ac ions. WT1 is in ol ed in he de elopmen o a numbe o o gans, including kidneys and gonads, spleen, ad enals, li e and diaph agm (Has ie, 2017). F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 1July 2021 | Volume 9 | A icle 683861 cell-09-683861 July 17, 2021 Time: 18:39 # 2 Díaz del Mo al e al. WT1 in Hea De elopmen Sys emic loss o unc ion o WT1 in mice causes emb yonic le hali y a midges a ion, a le hali y a ibu ed o de ec s in ca diac de elopmen (Ma ínez-Es ada e al., 2010;Cano e al., 2016). WT1 is highly exp essed in he emb yonic epica dium whe e i egula es a p ocess o epica dial-mesenchymal ans o ma ion and he de elopmen o he epica dial-de i ed cells ha con ibu e wi h ib oblas s and smoo h muscle o he ca diac connec i e and ascula issues (Ma ínez-Es ada e al., 2010;Von Gise e al., 2011). Fu he mo e, he epica dium is a sou ce o signals o myoca dial p oli e a ion and he de elopmen o he compac en icula wall. The pheno ype o WT1 loss o unc ion in mice in ol es hinning o he myoca dial walls associa ed o a de ec i e co ona y de elopmen , he mos p obable cause o he emb yonic dea h (La ine e al., 2005; Vega-He nández e al., 2011). The ca diac pheno ype o WT1 loss o unc ion had hi he o been exclusi ely ela ed wi h i s p ominen epica dial exp ession. Howe e , we ha e collec ed expe imen al e idence sugges ing ha WT1 is also exp essed a lowe le els in a ac ion o he emb yonic ca diomyocy es. This myoca dial exp ession o WT1 is hi he o poo ly known, and men ioned jus in a ew pape s (Villa del Campo e al., 2016;Van Ei e al., 2020; Wagne e al., 2021). Ruda and Kispe (2012) did no exclude a weak myoca dial exp ession o WT1 a E9.5 using he W 1TM1(EGFP/c e)W p d i e . Velecela e al. (2019) desc ibed a popula ion o cells in he emb yonic hea exp essing low le els o he epo e GFP in a W 1GFP knockin mouse model. This popula ion exp essed a numbe o myoca dial genes bu he au ho s conclude ha u he esea ch was needed in o de o cha ac e ize he na u e o his popula ion o cells p esumably exp essing WT1. A be e cha ac e iza ion o his p esump i e myoca dial exp ession o WT1 is also ele an since a lineage o ca diomyocy es de i ed om WT1-exp essing cells was conside ed as a ising om epica dial p ogeni o s (Zhou e al., 2008), gi ing ise o a con o e sy abou he myoca diogenic po en ial o epica dial-de i ed cells (Cai e al., 2008;Ruda and Kispe , 2012). The aim o his epo is o s udy in dep h he myoca dial exp ession o WT1 and o in es iga e i condi ional dele ion o WT1 in emb yonic ca diomyocy es causes al e a ions in ca diac de elopmen , anomalies ha could ha e been hi he o masked in p e ious desc ip ions o sys emic o epica dial-speci ic loss o unc ion. MATERIALS AND METHODS Animal Models The animals used in ou esea ch p og am we e handled in compliance wi h he ins i u ional and Eu opean Union guidelines (Di ec i e 2010/63/EU o he Eu opean Pa liamen ) o animal ca e and wel a e. The p ocedu es used in his s udy we e app o ed by he Commi ee on E hics o Animal Expe imen s o he Uni e si y o Málaga (p ocedu e code 2018-0018). Acco ding o hese p ocedu es, he mice we e eu hanized by ce ical disloca ion. Fo elec oca diog aphy, animals we e anes he ized wi h Ke amine, 64 µg/g body weigh and Xylazine 10 µg/g in PBS (i.p.). Fo ca diac MRI, animals we e anes he ized wi h a 1% iso lu ane in medical ai a a low a e o 1 L/min in an induc ion chambe and hen ans e ed o he MRI c adle. Respi a o y a e was moni o ed h oughou he MRI expe imen and he anes hesia low was eadjus ed when needed o main ain a a e o 60 ±10 bpm. The WT1GFP knockin line (Hosen e al., 2007) in which he exon 1 o a W 1 allele has been eplaced by he GFP sequence was used as a epo e o ac i e WT1 ansc ip ion. The Tg(WT1-c e)#Jbeb mouse line (WT1C e om now on) has been used in p e ious s udies o ace he WT1-exp essing cell lineage o o dele e speci ic genes in WT1-exp essing cells (Del Mon e e al., 2011;Wessels e al., 2012;Cano e al., 2013, 2016; Casano a e al., 2013). The gene a ion o G2-GATA4C e line has been desc ibed elsewhe e (Rojas e al., 2005). This d i e allows condi ional dele ion o genes in he la e al mesode m including p oepica dium and epica dium (Cano e al., 2016). Fo ca diac oponin T (Tnn 2 om now on) lineage acing and condi ional dele ion o WT1 in ca diomyocy es, we ha e used he Tg(Tnn 2-c e)5Blh mouse s ain. The Tnn 2 p omo e d i es ea ly exp ession o C e ecombinase in he ca diomyocy e lineage beginning a E7.5 (Jiao e al., 2003). We ha e also desc ibed he ac i a ion o his d i e and i s ecombina ion in a ac ion o he p oepica dial cells (Ca mona e al., 2020) as desc ibed below. C ossing his s ain wi h he W 1LoxP mice [desc ibed in Ma ínez-Es ada e al. (2010)] causes WT1 dele ion in he Tnn 2-exp essing cell lineage. In he con ex o his pape , we will call “mu an ” o all he mice wi h a Tnn 2C e/+;W1 lox/ lox geno ype. Fo lineage acing s udies, he mu ine lines ca ying he WT1C e and he Tnn 2C e d i e s we e c ossed wi h homozygo e Rosa26EYFP [B6.129 ×1-G (ROSA)26So m1(EYFP)Cos/J] mice (S ini as e al., 2001) o gene a e pe manen epo e exp ession in he lineages o WT1 and Tnn 2-exp essing cells. Emb yos we e s aged om he ime poin o aginal plug obse a ion, which was designa ed as he s age E0.5. Whole emb yos we e excised om u e us, washed in PBS and dissec ed o ob ain he hea s o ixed in 4% esh pa a o maldehyde solu ion in PBS o 2–8 h. Fixed emb yos we e pa a in-embedded o washed in PBS. Adul hea s we e ixed o 4–6 h ollowing he same p ocedu e. Analy ical Flow Cy ome y Fo low cy ome y analysis, dissec ed hea s we e dissocia ed o 20 min a 37◦C in p e-wa med 0.1% collagenase (C9407, Sigma) solu ion in PBS and homogenized by epea ed pipe ing. Cell suspensions we e washed in cy ome y bu e (PBS plus 2% e al bo ine se um and 10 mM HEPES) and il e ed h ough a 70 µm nylon mesh. Then, cells we e incuba ed on ice in he da k wi h he luo och ome-conjuga ed an ibodies. DAPI s aining showed ha less han 10% o he cells we e damaged. Ca diomyocy es o E10.5–E15.5 emb yos we e iden i ied by VCAM-1 immunos aining, since his cell adhesion molecule is only exp essed by he myoca dium in ea ly de elopmen al s ages (Pon én e al., 2013, see also Supplemen a y Figu e 1). Ca diomyocy es om E15.5–E17.5 emb yos we e iden i ied using a mi ochond ial s aining eagen (Mi o acke Deep Red, F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 2July 2021 | Volume 9 | A icle 683861 cell-09-683861 July 17, 2021 Time: 18:39 # 3 Díaz del Mo al e al. WT1 in Hea De elopmen In i ogen, M22426) (Ha o i e al., 2010). We incuba ed he cell suspension in he Mi o acke solu ion (100 nM in cy ome y bu e ) a 37◦C o 30 min. Cells we e analyzed in a FACS Ve se low cy ome e . Da a we e analyzed wi h Kaluza Analysis so wa e ( e sion 2.1) and displayed on ables as mean ±s anda d e o o mean. Nega i e con ols (C e-nega i e li e ma es) and iso ypic an ibodies allowed se ing o he ga es. De ails o he an ibodies used o low cy ome y a e p o ided in Supplemen a y Table 1. Cell Cul u e Hea s om E10.5 wild ype emb yos we e dissec ed and minced in s e ile Ca2+and Mg2+- ee PBS wi h 20 mM o 2,3- bu anedione 2-monoxime (2,3-BDM; Sigma #B0753). Then, he agmen s we e diges ed in Medium 199 wi h 20 mM o BDM and 0.2 mg/mL ypsin o 5 min a 37◦C. The cell suspension was washed wi h high glucose DMEM:M199 (0.7:0.3), plus 10% ho se se um, 0.5% FBS and penicillin/s ep omycin. The cells we e cul u ed wi h 5% CO2 o 48 h, and we e hen ixed in 4% pa a o maldehyde o 10 min and p ocessed o immuno luo escence. Immuno luo escence and Con ocal Mic oscopy Depa a inized sec ions o ixed cul u ed cells we e washed in T is-PBS (TPBS) and blocked o non-speci ic binding wi h SBT (16% sheep se um, 1% bo ine albumin, and 0.1% T i on X- 100 in TPBS). When bio inyla ed seconda y an ibodies we e used, endogenous bio in was blocked wi h he A idin-Bio in blocking ki om Vec o . Single immuno luo escence was pe o med incuba ing he sec ions wi h he p ima y an ibody o e nigh a 4◦C, washing in TPBS and incuba ing wi h he co esponding luo och ome-conjuga ed seconda y an ibody o 1 h a oom empe a u e. Double immuno luo escence was pe o med by mixing bo h p ima y an ibodies ( abbi polyclonal and mouse/ a monoclonal), and incuba ing o e nigh a 4◦C. We hen used a bio in-conjuga ed seconda y an ibody, ollowed by 45 min incuba ion wi h Cy5-conjuga ed s ep a idin. Nuclei we e coun e s ained wi h DAPI (Sigma, D9542). Sec ions we e moun ed in PBS:Glyce ol 1:1. De ails o he an ibodies used o immuno luo escence a e p o ided in Supplemen a y Table 1. Analysis o T abecula ion and P oli e a ion His ological sec ions o E13.5 mu an and con ol hea s we e used o assess he a e o p oli e a ion and he ela i e hickness o he compac and he abecula ed en icula laye s. Fo cell p oli e a ion, p egnan emales we e in ape i oneally injec ed wi h 3 mg o B dU (B5002, Sigma) dilu ed in 300 µL o PBS. Two hou s a e he injec ion he mice we e eu hanized and he emb yos we e p ocessed o B dU immuno luo escence as desc ibed abo e. Con ocal images we e analyzed by coun ing he numbe o B dU+ cells in he sec ion and measu ing he o al a ea o he nuclei s ained wi h DAPI (1/1000) using ImageJ so wa e. The mean a ea o he nuclei was es ima ed, and he o al numbe o nuclei in he sec ion was es ima ed di iding he o al DAPI s ained a ea by he mean a ea o he nuclei. Pe cen ages o B dU+ nuclei we e calcula ed o each issue sec ion. We analyzed 29 sec ions o six wild ype and 15 sec ions o h ee mu an emb yos. The deg ee o en icula compac ion was es ima ed by di iding he hickness o he compac laye by he o al hickness o he ca diac wall (compac plus abecula ed), in he midpoin be ween he base and he apex o he en icle. We analyzed 40 his ological sec ions om 12 con ol and 18 mu an E13.5 emb yos, and 5 con ol and 5 mu an E15.5 emb yos. ECG and MRI Elec oca diog ams we e ob ained om anes he ized mice using he da a acquisi ion sys em BioAmp Powe Lab 8/35 (ADIns umen s). Nega i e and posi i e elec odes we e placed in he uppe igh and lowe le limbs, espec i ely, and he neu al pole was placed in he lowe igh limb. We analyzed a sec ion o he ECG (abou 1 min) using he ECG module o he LabCha 8.0 so wa e and he p ese mouse se ings. MRI expe imen s we e ca ied on a B uke BioSpec MRI sys em (B uke BioSpec, B uke BioSpin, E lingen, Ge many) equipped wi h 400 mT m-1 ield g adien s and a 40 mm quad a u e bi d-cage esona o . Cine sho -axis (SAX) and long-axis (LAX) we e acqui ed using a e ospec i e sel -ga ed FLASH sequences (In aGa e, B uke BioSpin MRI, E lingen, Ge many). The acquisi ion pa ame e s we e as ollows: α= 20◦, TE = 2.88 ms, TR = 8.5 ms, FOV = 11 ×22 mm, ma ix size = 192 ×384, mo ie ames = 16, numbe o slices = 1, and slice hickness = 0.8. qPCR To al RNA was ex ac ed om whole hea s o Tnn 2C e+/−; W 1 lox/ lox (mu an ) and Tnn 2C e−/−; W 1 lox/ lox (con ol) E13.5 emb yos using NucleoSpin RNA XS (Mache ey Nagel) acco ding o he manu ac u e ’s ins uc ions and quan i ied by a Nanod op ND-1000 Spec opho ome e . Real ime PCR expe imen s we e pe o med wi h 20 ng o cDNA, SsoFas E aG een-mix (Bio-Rad, He cules. CA, Uni ed S a es) and co esponding p ime se s. qPCRs we e pe o med using a Ligh Cycle R 96 he mocycle (Roche). Gapdh and β-ac in we e used as in e nal con ols. Analysis o ela i e gene exp ession da a was pe o med using he 2−11CT me hod (Li ak and Schmi gen, 2001). Each PCR eac ion was ca ied ou in iplica e and epea ed in a leas h ee dis inc biological samples (a whole E13.5 hea o sample) o ob ain ep esen a i e means. Sequences o he p ime s used o qPCR a e p o ided in Supplemen a y Table 2. RNASeq RNA-Seq analysis was pe o med on h ee wild ype (Tnn 2C e−/−; W 1 lox/ lox)and h ee mu an E13.5 emb yos. To al RNA om each hea was ex ac ed by using RNAqueousTM-Mic o To al RNA Isola ion Ki (Ambion, AM1931) ollowing manu ac u e ’s ins uc ions. T ansc ip ome F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 3July 2021 | Volume 9 | A icle 683861 cell-09-683861 July 17, 2021 Time: 18:39 # 4 Díaz del Mo al e al. WT1 in Hea De elopmen analysis o o al RNA was pe o med by No ogene1. Only RNA samples wi h RNA in eg i y numbe (RIN) highe han 8.5 we e selec ed o lib a ies p epa a ion. 200 ng o o al RNA was subsequen ly used o p epa e RNA-Seq lib a y by using T uSeq RNA sample p ep ki (Illumina) acco ding o manu ac u e ’s ins uc ions. Pai ed-end (2 ×150) RNA sequencing was pe o med on Illumina No aSeq 6000 pla o m (Illumina). Fo imming and alignmen o aw da a, as q sequences eads we e uploaded o he Eu opean e sion o Galaxy pla o m (A gan e al., 2016). Reads we e immed wi h T im Galo e so wa e (Galaxy Ve sion 0.4.3.1) and aligned o he buil - in mouse e e ence genome mm10 wi h RNA STAR Gapped- ead mappe o RNA-seq da a (Galaxy Ve sion 2.6.0b-2) (Dobin e al., 2013). Fo gene exp ession analysis, bam iles we e downloaded om Galaxy se e and analyzed wi h di e en RS udio e sion 1.1463, R e sion 3.4.4 packages downloaded om Bioconduc o websi e2. Reads we e assigned o genes by using “ ea u eCoun s” unc ion om package “Rsub ead” (Liao e al., 2019) e sion 1.28.1 and mouse anno a ion ile elease M19, GRCm38.p6. Uniquely, mapped eads we e used o calcula e gene exp ession. Di e en ial gene exp ession analysis was pe o med h ough package “DESeq2” e sion 1.26.0 (Lo e e al., 2014). All gene compa ison wi h padj <0.05 (Benjamini & Hochbe g es ) we e conside ed di e en ially exp essed among expe imen al condi ions. GO Func ional En ichmen Analysis was pe o med h ough O e Rep esen a ion Analysis (ORA) Me hod (Boyle e al., 2004) by using “clus e P o ile ” .3.6.0. package (Yu e al., 2012). Gene se s wi h p- alue <0.05 we e conside ed o e - ep esen ed among expe imen al condi ions. S a is ics S a is ical compa ison o means was pe o med using wo- ailed S uden ’s – es when he da a we e no mally dis ibu ed (Kolmogo o -Smi no es ). Mann-Whi ney U- es was used when da a di e ed signi ican ly om a no mal dis ibu ion. A Chi-squa e es was used o compa ison be ween obse ed and expec ed equencies o mu an s. We conside ed p<0.05 as he c i e ion o s a is ical signi icance. RESULTS WT1 Is Exp essed in a F ac ion o Ca diomyocy es Du ing De elopmen The W 1GFP knockin mouse model (he e ozygo e, GFP/ + emb yos) allowed us o epo ac i a ion o he W 1 locus. Using analy ical low cy ome y we ha e ound ha a ac ion o he non-endo helial cells o he de eloping hea shows some deg ee o GFP exp ession (Figu e 1). Epica dial cells can be easily ecognized by hei s ong GFP exp ession (Velecela e al., 2019). Myoca dial cells o he ea ly de elopmen al s ages can be sa ely iden i ied by VCAM immuno eac i i y, absen in endoca dial and epica dial cells (Pon én e al., 1h ps://en.no ogene.com/ 2h p://bioconduc o .o g 2013, see also Supplemen a y Figu e 1). A ac ion o he VCAM+ ca diomyocy es (abou 13% o he non-endo helial cells) exp ess low le els o GFP a E10.5 (Table 1). This pe cen age dec eases a E14.5–E15.5 (Figu e 1A and Table 1). As he myoca dium educes i s GFP exp ession, a popula ion o epica dial-de i ed mesenchymal cells (EPDC) appea s and p og essi ely acqui es VCAM immuno eac i i y (Figu e 1A and Supplemen a y Figu e 2). Due o his eme ging VCAM exp ession in non-myoca dial cells, we iden i ied ca diomyocy es a la e s ages by hei mi ochond ial con en (Mi o acke s aining, Ha o i e al., 2010) and we con i med he dec ease in he myoca dial exp ession o WT1. Only a small ac ion o Mi o acke -posi i e ca diomyocy es (2% in he en icles, 4% in he a ia) showed ac i a ion o he WT1 epo e a E17.5 (Figu e 1B and Table 1). We u he con i med he p esence o WT1 p o ein in ca diomyocy es by immunocy ochemis y in bo h, cul u ed E10.5 emb yonic ca diomyocy es and his ological sec ions (Figu es 2A,B and Supplemen a y Figu e 3). GFP+ ca diomyocy es can be seen sca e ed h oughou he hea in he e ozygous E9.5–E11.5 W 1GFP/+emb yos (Figu es 2C,D). Exp ession o he epo e indica es W 1 p omo e ac i a ion in hese cTnT+ ca diomyocy es, Myoca dial immunolocaliza ion o WT1 becomes es ic ed o he sinus enosus in E12.5 emb yos (Figu es 2E–G), mainly i i s pos e io wall. The WT1C e;R26REYFP lineage acing sys em allowed us o es ablish he dis ibu ion o he ca diomyocy e lineage whe e he W 1 p omo e was ac i a ed. As shown in Figu es 2H–J, hese WT1-lineage ca diomyocy es can be seen h oughou he hea , wi h a g ea e abundance in he sinus enosus, le en icle and le pa o he in e en icula sep um. WT1-lineage ca diomyocy es can also be obse ed in he en icles o adul hea s (Figu e 2K). We quan i ied he equency o WT1-lineage ca diomyocy es by analy ical low cy ome y. A E12.5 and E15.5 abou 18 and 25% o he ca diomyocy es show exp ession o he epo e , espec i ely (Figu e 3 and Table 1). The la e pe cen age emains b oadly cons an in la e emb yos (no shown). In summa y, he W 1 p omo e is ansien ly ac i a ed in he lineage o 20–25% o he emb yonic ca diomyocy es, and WT1 p o ein can be de ec ed by immunocy ochemis y in a ac ion o hese ca diomyocy es, mainly in ea ly de elopmen al s ages. Myoca dial Dele ion o WT1 Causes S uc u al De ec s in he Hea The Tnn 2C e d i e ac i a es C e-media ed ecombina ion in i ually all he myoca dium a ea ly de elopmen al s ages (Jiao e al., 2003). When we checked he e iciency o his ecombina ion in he Tnn 2C e;R26RYFP model, we ound ha a small ac ion o he epica dial cells a ea ly s ages exp ess he YFP epo e , sugges ing de i a ion om Tnn 2-exp essing p ogeni o s (Figu e 2B). We ha e es ima ed elsewhe e ha abou 30% o he ea ly emb yonic epica dium (E11.5) de i e om a Tnn 2 exp essing lineage (Ca mona e al., 2020). In he p esen s udy we ound ha 43 ±11% o he epica dial cells de i e F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 4July 2021 | Volume 9 | A icle 683861 cell-09-683861 July 17, 2021 Time: 18:39 # 5 Díaz del Mo al e al. WT1 in Hea De elopmen FIGURE 1 | Exp ession o he GFP epo e in ca diomyocy es o he WT1GFP/+knockin line (GFP/ + = he e ozygous emb yos; c l + / + = homozygous nega i e con ols). Rep esen a i e cy og ams ob ained om he analysis o 29 emb yos. (A) Ca diomyocy es can be iden i ied as VCAM+ /CD31- cells in he ea lies s ages o ca diac de elopmen . A ac ion o hese ca diomyocy es exp esses he GFP epo e ( op ow) a lowe le els han epica dial cells. In s ages E13.5–E15.5 he epo e exp ession dec eases in he VCAM + popula ion. No e he inc easing popula ion o VCAM + exp essing cells ha ep esen he epica dial-de i ed mesenchymal cells (EPDC). (B) S age E17.5. Ca diomyocy es can be iden i ied by Mi o acke s aining in his la e de elopmen al s age. A small ac ion o a ial and en icula ca diomyocy es s ill exp esses he GFP epo e . TABLE 1 | Pe cen age o ca diomyocy es (de ined as indica ed in he pheno ype column) exp essing he epo e GFP (knockin line W 1GFP/+) o YFP (lineage acing line W 1C e; R26REYFP) (mean ±s.e.m.). Model S age Pheno ype % Ca diomyocy es N WT1GFP E10.5 CD31-/VCAM + /GFPlow 13.5±1.2 4 E11.5 CD31-/VCAM + /GFPlow 13.7±1.6 4 E13.5 CD31-/VCAM + /GFPlow 10.8±0.7 4 E14.5 CD31-/VCAM + /GFPlow 7.6±0.6 5 E15.5 CD31-/VCAM + /GFPlow 3.5±0.6 4 E17.5 A ia CD31-/Mi o acke high/GFPlow 4.2±0.6 4 E17.5 Ven icles CD31-/Mi o acke high/GFPlow 2.2±0.4 4 WT1C e;R26REYFP E12.5 CD31-/VCAM + /YFP + 17.7±5.1 4 E15.5 A ia CD31-/Mi o acke high/YFP + 23.6±7.0 3 E15.5 Ven icles CD31-/Mi o acke high/YFP + 26.4±4.4 4 om a Tnn 2-exp essing lineage a he s age E13.5 (mean o h ee emb yos). Thus, he condi ional dele ion o WT1 in he Tnn 2 lineage should dele e WT1 in all he myoca dium, bu only in a ac ion o he epica dium and he epica dial-de i ed mesenchymal cells du ing he p ocess o epi helial-mesenchymal ansi ion. This ac ion mus be be ween 30–40%, since his is he pe cen age o Tnn 2-lineage epica dial cells when he epi helial-mesenchymal ansi ion is a i s peak (i.e., be ween he s ages E10.5 and E13.5). We did no de ec signi ican emb yonic le hali y p o oked by his dele ion, al hough he pe cen age o adul mu an mice signi ican ly dec eased om he expec ed 25% o a 13.7% The pe cen age o neona al mice was also educed (12.8%), sugges ing ha some mo ali y migh occu pe ina ally (Table 2). F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 5July 2021 | Volume 9 | A icle 683861 cell-09-683861 July 17, 2021 Time: 18:39 # 6 Díaz del Mo al e al. WT1 in Hea De elopmen FIGURE 2 | WT1 exp ession in ca diomyocy es. Rep esen a i e images ob ained om he analysis o >30 emb yos. (A) Immunolocaliza ion o p o ein WT1 in cul u ed ca diomyocy es cha ac e ized by ca diac oponin exp ession (a ow) and ob ained om an E10.5 emb yo. (B) Immunolocaliza ion o p o ein WT1 in an E11.5 Tnn 2C e;EYFP emb yo. WT1 ( ed s aining) is localized in some ca diomyocy es (inse s). Epica dial cells (EP) show exp ession o he p o ein WT1, and a ew de i e om a ca diac oponin-exp essing lineage (yellow a ows). RA: igh a ium; LV, RV: le and igh en icle, espec i ely. (C,D) WT1GFP/+emb yos, E9.5 and E11.5, espec i ely. Exp ession o he epo e e eals WT1 p omo e ac i a ion in a numbe o ca diomyocy es o hese ea ly emb yos. Colocaliza ion wi h ca diac oponin (cTnT) is shown by a ows. The inse s show he sepa a e GFP channel. Inse in (D) shows some GFP-exp essing ca diomyocy es loca ed in he wall o he igh sinus enosus (a owhead). LA, LV: le a ium and en icle; PE: p oepica dium; LSV, RSV: (Con inued) FIGURE 2 | Con inued le and igh sinus enosus. (E–G) Exp ession o WT1 p o ein in he walls o he igh and le sinus enosus (RSV, LSV) o an E12.5 cTnn 2C e;EYFP emb yo. A ows show posi i e nuclei o ca diomyocy es iden i ied by he ca diac oponin lineage epo e YFP. Figu e (G) shows a highe magni ica ion o he le sinus enosus wall in he ed (WT1) channel. A s onge exp ession is de ec ed in he epica dial cells (EP). (H–J) Exp ession o he WT1 lineage epo e in he WT1C e;EYFP line, s ages 11.5, 15.5, and 19.5, espec i ely. Colocaliza ion wi h smoo h muscle ac in is shown in he pos e io wall o he igh sinus enosus (a ow in H), and i becomes equen in he en icles in la e s ages, pa icula ly in he le pa o he in e en icula sep um (IVS). LCCV: le common ca dinal ein. (K) Exp ession o he WT1 lineage epo e in an adul WT1C e;EYFP hea . A numbe o ca diomyocy es show epo e exp ession. Scales: 50 µm, excep (D,H,I) (100 µm) and, (J) (200 µm). All mu an emb yonic hea s, om E13.5 onwa d, showed i egula shapes, hin ee en icula walls oge he wi h high deg ee o abecula ion, a ial pec ina e muscles less de eloped o absen , and de ec i e sinus enosus (Figu es 4A– D,5). In 8 ou o 14 emb yos analyzed (57%) we also obse ed s uc u al de ec s in he en icula and a ial walls (Figu es 4E–K). Namely, in wo emb yos (14%) he a ial wall was discon inuous and communica ed wi h he en icula ca i y (Figu e 4E) o wi h a subepica dial ein (Figu es 4G,H). Six emb yos analyzed (43%) showed muscula de ec s in he in e en icula sep um (Figu es 4E,J), discon inui ies (Figu e 4F) o di e icula in he en icula walls (Figu e 4K). Despi e hese myoca dial abno mali ies, he co ona y ascula u e de eloped no mally and co ona y a e ies we e obse ed in la e mu an emb yos and adul mu an mice (Supplemen a y Figu e 4). The o igin o he co ona y a e ies om he ao a showed a no mal pa e n in mu an mice, wi hin he ana omical a iabili y o his ea u e (Fe nández e al., 2008). We measu ed he a io compac / o al myoca dial hickness in E13.5 and E15.5 emb yos (Table 3). The en icula compac laye was hinne in mu an emb yos as compa ed wi h he o al myoca dial hickness. The di e ence was signi ican in he le en icle o he E13.5 emb yos and in bo h en icles o he E15.5 emb yos. P oli e a ion o ca diac cells in con ol and mu an E13.5 emb yos was analyzed by es ima ing he pe cen age o B dU+ cells (Table 3). The pe cen age o p oli e a ing cells was signi ican ly educed in he mu an a ia and i also showed a lowe alue in he en icles, al hough he di e ence wi h con ols was no signi ican . We compa ed he pheno ype o he dele ion o WT1 in he ca diac oponin lineage wi h ha om o he wo mu ine models o WT1 loss o unc ion, a la e al mesode m/p oepica dial/epica dial condi ional dele ion (G2-GATA4C e;WT1 lox,Cano e al., 2016) and a sys emic dele ion in all he emb yonic issues (homozygous knockin WT1GFP/GFP,Hosen e al., 2007). Bo h dele ions a e le hal a midges a ion. The hea wi h p o/epica dial dele ion o WT1 showed lack o co ona y a e ies, bu no mal de elopmen o he in e en icula sep um and pec ina e muscles (Figu es 4L,M). F ee myoca dial walls ne e showed discon inui ies. Howe e , he emb yos wi h sys emic WT1 loss o unc ion showed de ec s F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 6July 2021 | Volume 9 | A icle 683861 cell-09-683861 July 17, 2021 Time: 18:39 # 7 Díaz del Mo al e al. WT1 in Hea De elopmen FIGURE 3 | Exp ession o he YFP epo e in ca diomyocy es o he WT1C e; R26REYFP lineage acing line. Rep esen a i e cy og ams ob ained om he analysis o 11 emb yos. The ac ion o ca diomyocy es exp essing he WT1 lineage epo e (YFP+) is shown. TABLE 2 | F equency o geno ypes by age. To al Wild He e ozygo es Mu an s % Mu <E12.5 52 24 15 13 25.00 E13.5–E15.5 375 204 87 84 22.40 E16.5–E19.5 34 16 8 10 29.41 Neona es 39 21 13 5 12.82† Adul s 131 67 46 18 13.74* To al 631 332 169 130 20.60 *Di e ence be ween he obse ed equency and he expec ed one (25%) is signi ican (p <0.05, Chi-squa e es ). †No signi ican di e ence (p <0.08, Chi-squa e es ). in he in e en icula sep um simila o hose displayed by he Tnn 2C e;WT1 lox mu an emb yos (Figu e 4N). As s a ed abo e, bo h sinus enosus ho ns we e abno mal in he mu an emb yos (Figu e 5). The le sinus enosus was embedded in o he le pleu ope ica dial memb ane, while he igh sinus was also abno mally connec ed wi h he igh pleu ope ica dial memb ane. In bo h cases, he pos e io walls o he sinus ho ns in mu an s we e limi ed by he pleu al ca i ies, while he sinus ho ns in con ol mice we e su ounded by he pe ica dial ca i y (Figu es 5A,B). The igh sinus ho n lacked myoca dium in i s pos e io pa (as e isks in Figu e 5E), a egion ha is muscula in he con ols o he same s age (Figu es 5D,H). Due o he lack o muscle in his a ea, mos o he HCN4 exp ession was localized in he wall o he igh a ium (Figu es 5E,I). This abno mal pa e n o HCN4 exp ession was also obse ed in he a ea co esponding o he sinoa ial node in E18.5 emb yos, an a ea ha was much smalle in mu an s (Supplemen a y Figu e 5). The myoca dial laye was e y hin on he pos e io pa o he le sinus enosus (Figu es 5G,K). Bo h, he sys emic dele ion o WT1 (No den e al., 2010) and he la e al mesode m/p oepica dial/epica dial-speci ic dele ion o WT1 using he G2 enhance o he GATA4 gene (Cano e al., 2016) showed a mo e se e e pheno ype, wi h lack o pleu ope ica dial memb anes and comple e loss o he pos e io walls o he sinus enosus, which does no o m clea ly de ined ho ns (as e isk in Figu e 5C). Adul Mice Wi h Myoca dial Dele ion o WT1 Show Anomalies in Ca diac S uc u e and Func ion As s a ed abo e, mu an mice a e iable al hough hei equency is abou a hal o he expec ed in he adul popula ion. In adul mu an mice, he hea weigh wi h espec o o al body F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 7July 2021 | Volume 9 | A icle 683861 cell-09-683861 July 17, 2021 Time: 18:39 # 8 Díaz del Mo al e al. WT1 in Hea De elopmen FIGURE 4 | Emb yonic pheno ype o he condi ional WT1 loss o unc ion in ca diomyocy es (Tnn 2C e/+; WT1 lox/ lox line). Rep esen a i e images ob ained om he analysis o 14 mu an emb yos. (A–D) Gene al iew o he hea in con ol (A,C) and mu an (B,D) emb yos, s ages E13.5 and E14.5. Mu an emb yos show hinne en icula walls, de ec i e sinus enosus (shown in mo e de ail in Figu e 5) and lack o pec ina e muscles (PM) in he igh a ium (RA). RV: igh en icle; LA: le a ium; LV: le en icle; RSV: igh sinus enosus; LSV: le sinus enosus. (E–H) De ec s in he myoca dial walls and in e en icula sep um (IVS) ound in mu an emb yos. These de ec s include abno mal a io en icula connec ion and muscula IVS de ec (a ow and a owheads in E), discon inui ies in he igh en icula (RV) ee wall (a ows in F) and connec ion o he le a ial (LA) lumen wi h a subepica dial co ona y ein (CV) (G,H).(I,J) IVS de ec (a ow), hin compac myoca dium and hype abecula ion (as e isks) in an E19.5 mu an emb yo (J) compa ed wi h a con ol o he same age (I).(K) The lack o compac ion o he le en icula apex causes a di e iculum p o uding in he su ace o he hea (a ows). No e he lack o compac myoca dium a he base o he IVS. (L,M) Rep esen a i e E15.5 emb yo o he G2-GATA4C e;WT1 lox model o condi ional dele ion o WT1 in la e al mesode m, including sep um ans e sum and p o/epica dium (Cano e al., 2016). The main ea u es o his pheno ype a e he de ec i e sinus enosus and epica dium (EP), bu he de elopmen o he pec ina e muscles (PM) and he in e en icula sep um (IVS) a e no mal. (N) Sys emic dele ion o WT1 (homozygous knockin W 1GFP/GFP,Hosen e al., 2007), E14.5, espec i ely. This is he mos ad anced s age eached by hese mu an emb yos. No e he de ec s in he IVS (a ows). Scales: 50 µm and excep (C–E) (100 µm). weigh was signi ican ly educed (0.54 ±0.06% in mu an s s. 0.84 ±0.11% in con ols, N= 9 and 6, espec i ely, p<0.05). The hea showed a highly i egula shape (Figu e 6A), lacking pec ina e muscles in he igh a ium, which appea ed ound- shaped (Figu es 6A,C,D). In a case, he le en icle was pa ially di ided by an ec opic muscula sep um (Figu e 6B). One o he le en icula ca i ies ended in an aneu ism p o uding in he base o he le en icle (Figu e 6E). A eas o ib osis, diso ganiza ion o he myoca dium and di e icula we e equen ly obse ed in he ca diac walls (Figu es 6B,F–H). In i o MRI images con i med he anomalies o he igh a ium and he le en icula wall [Figu es 6I–K and Supplemen a y Videos 1 (con ol) and 2(mu an )]. Adul mu an mice also showed al e a ions in he ECG, wi h signi ican inc ease o he RR in e al and dec ease o he PR in e al as compa ed o con ols (p<0.05, S uden ’s - es ). The QRS in e al was also signi ican ly longe in he mu an s (16.3 s. 14.4 ms in he con ols, p<0.05). On he o he hand, he ampli udes o he P and R wa es, and he heigh o he S-T in e al (co esponding o he J wa e, see sec ion “Discussion”) we e signi ican ly educed in mu an mice (Table 4 and Supplemen a y Figu e 6). F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 8July 2021 | Volume 9 | A icle 683861 cell-09-683861 July 17, 2021 Time: 18:39 # 9 Díaz del Mo al e al. WT1 in Hea De elopmen TABLE 3 | P oli e a ion index (% o B dU+ nuclei) and en icula compac ion index (% compac laye hickness ela ed o o al wall hickness) in condi ional WT1 mu an and con ol mice (s ages E13.5 and E15.5). S age E13.5 S age E15.5 P oli e a ion Iindex (%B dU+ cells) A ium con ol 8,0.35 ±1,0.04 (N= 11) p<0,0.013† A ium mu an 3,0.46 ±1,0.10 (N= 5) Ven icle con ol 5,0.45 ±0,0.71 (N= 18) p<0,0.290 (n.s.) Ven icle mu an 4,0.12 ±1,0.03 (N= 9) Ven icula compac ion index (% compac laye hickness ela ed o o al wall hickness) Righ en icle con ol 23,0.8 ±3,0.2 (N= 13) p<0,0.102 (n.s.) 58,0.4 ±5,0.0 (N= 7) p<0,0.013* Righ en icle mu an 18,0.4 ±1,0.5 (N= 19) 40,0.0 ±3,0.9 (N= 7) Le en icle con ol 24,0.6 ±2,0.6 (N= 13) p<0,0.0005* 53,0.5 ±2,0.5 (N= 8) p<0,0.002* Le en icle mu an 14,0.4 ±1,0.2 (N= 19) 41,0.2±2,0.1 (N= 7) N is he numbe o sec ions analyzed. 6 con ol and 3 mu an emb yos we e used o he p oli e a ion s udy. Fo he en icula compac ion index we analyzed 12 con ol and 18 mu an E13.5 emb yos, and 5 con ol and 5 mu an E15.5 emb yos. n.s: no signi ican . †Mann Whi ney U- es . *S uden ’s - es . FIGURE 5 | Sinus enosus de ec s caused by he o he condi ional WT1 loss o unc ion in ca diomyocy es (Tnn 2C e/+; WT1 lox/ lox line). (A,B) Compa ison o he igh and le sinus enosus (RSV, LSV) in E13.5 con ol and mu an emb yos, espec i ely. The sinus enosus in he con ol emb yos show a pos e io wall co e ed by he epica dium, limi ed by he pe ica dial ca i y (PC) and ee om he pleu ope ica dial memb anes (PPC). The mu an emb yos show pos e io walls con inuous wi h he pleu ope ica dial memb anes and limi ed by he pleu al ca i ies (PL). (C) G2-GATA4C e;WT1loxP model o condi ional dele ion o WT1 in la e al mesode m. In his E13.5 mu an emb yo he PPC a e lacking, and he pos e io wall o he igh and le sinus enosus a e embedded in he body walls (as e isks). (D–K) Immunocy ochemical ea u es o he pos e io wall o he mu an sinus enosus (E,G,I,K) as compa ed wi h he con ol emb yos (D,F,H,J). S age E15.5. The mu an walls show ewe ca diomyocy es and myoca dial discon inui ies (as e isk in E, a ows in K, compa e wi h a ows in D,H,J). No e he lack o HCN4 exp ession in he pos e io wall o he mu an igh sinus enosus (as e isks in E,I). The exp ession o HCN4 is ex ended in hese emb yos owa d he igh a ial wall (RAW). Scales: 50 µm. T ansc ip omic Analysis o he Mu an Hea s Re eals Al e a ions in he Exp ession o Genes Rela ed o Myoca dial Func ion RNASeq analysis was pe o med on whole E13.5 hea s. This is he s age whe e he main mo phological ea u es o he mu an pheno ype i s appea . A o al numbe o 137 genes we e di e en ially exp essed in he mu an hea as compa ed wi h con ols (adjus ed p- alues <0.05). 80 o hem we e up egula ed and 57 we e down egula ed (Supplemen a y Table 3 and Figu e 7). GO unc ional en ichmen analysis pe o med h ough o e ep esen a ion analysis me hod sugges ed ha he calcium ion egula ion was deeply al e ed in he mu an hea . In ac , h ee o he six main biological p ocesses a ec ed by he WT1 loss o unc ion we e associa ed o gene se s ela ed wi h calcium ion homeos asis, egula ion o cy osolic calcium concen a ion and calcium ion anspo . On he F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 9July 2021 | Volume 9 | A icle 683861 cell-09-683861 July 17, 2021 Time: 18:39 # 16 Díaz del Mo al e al. WT1 in Hea De elopmen No den, J., G ieskamp, T., Lausch, E., an Wijk, B., an den Ho , M. J., Engle , C., e al. (2010). W 1 and e inoic acid signaling in he subcoelomic mesenchyme con ol he de elopmen o he pleu ope ica dial memb anes and he sinus ho ns. Ci c. Res. 106, 1212–1220. doi: 10.1161/CIRCRESAHA.110.21 7455 Ohba, T., Wa anabe, H., Mu akami, M., Sa o, T., Ono, K., and I o, H. (2009). Essen ial ole o STIM1 in he de elopmen o ca diomyocy e hype ophy. Biochem. Biophys. Res. Commun. 389, 172–176. doi: 10.1016/j.bb c.2009.08.117 Ohlow, M. A. (2017). 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The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 16 July 2021 | Volume 9 | A icle 683861