Conditional deletion of WT1 in the septum transversum mesenchyme causes congenital diaphragmatic hernia in mice

*Fo co espondence: chapuli@ uma.es Compe ing in e es s: The au ho s decla e ha no compe ing in e es s exis . Funding: See page 14 Recei ed: 15 Ma ch 2016 Accep ed: 08 Sep embe 2016 Published: 19 Sep embe 2016 Re iewing edi o : Ma ga e Buckingham, Ins i u Pas eu , F ance Copy igh Ca mona e al. This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use and edis ibu ion p o ided ha he o iginal au ho and sou ce a e c edi ed. Condi ional dele ion o WT1 in he sep um ans e sum mesenchyme causes congeni al diaph agma ic he nia in mice Ri a Ca mona 1,2 , Ana Can ˜e e 1,2 , Elena Cano 3 , Lau a A iza 1,2 , Anabel Rojas 4,5 , Ramon Mun ˜oz-Cha ´puli 1,2 * 1 Depa men o Animal Biology, Uni e si y o Ma´laga, Ma´laga, Spain; 2 Andalusian Cen e o Nanomedicine and Bio echnology (BIONAND), Ma´laga, Spain; 3 Max Delb u€ck Cen e o Molecula Medicine, Be lin, Ge many; 4 Andalusian Cen e o Molecula Biology and Regene a i e Medicine (CABIMER), Se illa, Spain; 5 Cen o de In es igacio´ n Biome´ dica en Red de Diabe es y En e medades Me abo´ licas Asociadas (CIBERDEM), Se illa, Spain Abs ac Congeni al diaph agma ic he nia (CDH) is a se e e bi h de ec . W 1-null mouse emb yos de elop CDH bu he mechanisms egula ed by WT1 a e unknown. We ha e gene a ed a mu ine model wi h condi ional dele ion o WT1 in he la e al pla e mesode m, using he G2 enhance o he Ga a4 gene as a d i e . 80% o G2-Ga a4 C e ;W 1 l/ l emb yos de eloped ypical Bochdalek- ype CDH. We show ha he pos hepa ic mesenchymal pla e coelomic epi helium gi es ise o a mesenchyme ha popula es he pleu ope i oneal olds isola ing he pleu al ca i ies be o e he mig a ion o he somi ic myoblas s. This p ocess ails when W 1 is dele ed om his a ea. Mu an emb yos show Raldh2 down egula ion in he la e al mesode m, bu no in he in e media e mesode m. The mu an pheno ype was pa ially escued by e inoic acid ea men o he p egnan emales. Replacemen o in e media e by la e al mesode m ecapi ula es he e olu iona y o igin o he diaph agm in mammals. CDH migh hus be iewed as an e olu iona y a a ism. DOI: 10.7554/eLi e.16009.001 In oduc ion Congeni al diaph agma ic he nia (CDH) is a se e e bi h de ec , cha ac e ized by incomple e o ma- ion o muscula iza ion o he diaph agm and, as a consequence, he nia ion o he s omach, in es- ines, li e o spleen in o he pulmona y ca i ies, leading o pulmona y hypoplasia. CDH occu s app oxima ely in 1 ou o 3000 bi hs, accoun ing o abou 8% o all se e e congeni al anomalies. 80–90% o all he cases a e pos e ola e al he nias, also known as Bochdalek- ype CDH, cha ac e ized by a de ec in he pos e o (do sal in mice) la e al a ea o he diaph agm. In mos cases (>85%), his de ec is loca ed a he le side (Pobe e al., 2010). The classical hypo hesis is ha Bochdalek CDH is due o a ailu e o he usion o pleu ope i oneal olds (PPFs) wi h he sep um ans e sum (ST). PPFs a e la e al ims o issue connec ing caudally wi h he neph ic idges and an e io ly wi h he ST (Maye e al., 2011). Some au ho s desc ibe ha his usion a he occu s wi h he pos hepa ic mes- enchymal pla e (PHMP), an accumula ion o mesenchymal cells de i ed om he ST and loca ed in he pos e odo sal ma gin o he li e lobes (I i ani e al., 1984). Al hough all hese issues o m an ana omical con inuum, we will e e o he pos e io , do sola e al a eas o he li e as PHMP, and PPFs o he issue olds loca ed in he do sal pa o he coelomic ca i y, ma king he limi be ween he pe i oneal and he pleu al ca i ies. The clinical aspec s o CDH a e well s udied bu i s e iology is s ill poo ly known (Klaassens e al., 2006). Animal models a e he e o e e y aluable in o de o in es iga e he Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 1 o 17 RESEARCH ARTICLE cellula and molecula p ocesses leading o CDH in humans. Recen ly, i has been desc ibed in mice ha dele ion o GATA4 in he PPFs mesenchyme using a P x1 C e d i e leads o he de elopmen o amuscula , weak a eas in he diaph agm and CDH (Me ell e al., 2015). This pape assumes ha P x1 is exp essed in he mesenchyme o he pleu ope i oneal olds (PPFs) bu no in he ST, and in ac , i sugges s ha he ST con ibu es minimally o he de ini i e diaph agm. Howe e , his epo does no dis inguish be ween PPFs and PHMP and he pheno ype does no display diaph agma ic discon inui ies, nei he does i show a p e alence on he le side, which a e common ea u es in human CDH. The e inoic acid (RA) signaling pa hway has also been in ol ed in diaph agma ic de elopmen . A classical animal model o CDH consis s in he ea men o p egnan a s wi h ni o en, a subs ance ha inhibi s he syn hesis o RA. I has been shown ha ni o en ea men leads o educed size o he PPFs/PHMP, especially in he le side, dec easing cell p oli e a ion in his a ea (Clugs on and G een, 2007;Clugs on e al., 2010) and also dec easing exp ession o WT1 and GATA4 (Dingeman e al., 2011,2013). RA ea men in his model pa ially escues he pulmona y hypopla- sia de ec (Mon edonico e al., 2008;Sugimo o e al., 2008). The pheno ype o his animal model is hus, qui e simila o he human one. To da e, only a ew examples o CDH in human ha e been associa ed o mu a ions in WT1 locus, such as he Denys-D ash, Meacham and WAGR synd omes (Sco e al., 2005;Su i e al., 2007; An onius e al., 2008). WT1 is exp essed in he coelomic epi helium o he sep um ans e sum o mouse emb yos by he s age E9.0. Loss o unc ion o he Wilms’ umo supp esso gene W 1 in mice leads o de ec i e diaph agms (K eidbe g e al., 1993;Clugs on e al., 2006). Howe e , he mechanism by which WT1 con ibu es o diaph agm de elopmen is s ill unknown. To s udy he ole o WT1 in CDH we pe o med loss-o - unc ion expe imen s by condi ionally inac i a ing he W 1 gene in he ST/PHMP/PPFs mesenchyme. The use o WT1 condi ional knockou o e comes he ea ly emb yonic dea h caused by sys emic de iciency o WT1. We used a d i e based on he G2 enhance o he Ga a4 gene (Rojas e al., 2005). This enhance d i es exp ession o Ga a4 in he la e al pla e mesode m om he s age E7.5, and by he s age E9.5 is ac i e in he sep- um ans e sum and p oepica dium, ceasing i s ac i i y by E12.5 The ac i i y o his enhance is comple ely absen in he in e media e mesode m. Ou indings indica e ha WT1 is in ol ed in he gene a ion o he mesenchyme o he ST/PHMP/PPFs con inuum h ough epi helial-mesenchymal ansi ion and hey p o ide a no el pe spec i e on he genesis o he Bochdalek he nia and he e o- lu iona y o igin o he diaph agm. Resul s The sep um ans e sum, pos hepa ic mesenchymal pla e and pleu ope i oneal olds con ain he e ogeneous mesenchymal popula ions In no mal E10.5 emb yos, he pos e io and do sal ma gin o he li e shows an accumula ion o mes- enchymal issue, which ex ends om he do sal mesen e ium o he li e o he la e al ips o he lobes (Figu e 1). This mesenchymal laye is he PHMP desc ibed by I i ani (1984). The PPFs, by his de elopmen al s age, appea as a pai o ou g ow hs o he body wall loca ed a bo h sides o he lung buds. (Figu e 1A,B). They a e also cons i u ed o mesenchymal cells lined by he coelomic epi- helium. The G2-Ga a4 enhance di ec ed LacZ epo e exp ession in bo h PPF and PHMP a E10.5 and E11.5 in he mouse emb yo (Figu e 1A). Howe e , no be a galac osidase ac i i y was obse ed in he pos e io and medial pa o he sep um ans e sum, whe e he li e is connec ed wi h he diges i e ac (as e isk in Figu e 1A), indica ing ha he G2 enhance is no ac i e in his speci ic domain. PHMP and PPFs a e no ana omically independen en i ies. The la e al ips o he PHMP appea con inuous wi h he PPFs a he cephalic le el while hey a e sepa a ed mo e caudally (Figu e 1B). The con inuous pa o he PHMP/PPFs ensemble appa en ly o ms i sel by he an e io g ow h o he pe i oneal ecess, which sepa a es he li e sides om he body wall. Thus, he mesenchymal popula ion o he ST o igina es he PHMP and also he mos cephalic po ion o he PPFs. The la e al closu e o he pleu al ca i ies occu s be ween E11.5 and E12.5 by pos e io g ow h o he c escen - Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 2 o 17 Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine Figu e 1. Cell lineages in he sep um ans e sum (ST), pos hepa ic mesenchymal pla e (PHMP) and pleu ope i oneal olds (PPF). (A) G2-LacZ emb yo a he s age E11.5. The G2 enhance is ac i e in he li e (LI) meso helium and pos hepa ic mesenchymal pla e (PHMP). Howe e , he cen al a ea o he sep um ans e sum shows no ac i i y o his enhance (as e isk). (B)W 1 C e ;R26R LacZ emb yo, E10.5. Cells om he WT1 lineage appea in blue. Se ies o ans e se sec ions om cephalic (le ) o caudal ( igh ) le els. The an e io pe i oneal ecess (PR) is spli ing he li e om he body wall. The PHMP is con inuous wi h he PPF a cephalic le els. No e he abundance o mesenchymal cells in he an e io PHMP and how his mesenchyme is less abundan in pos e io le els. The PPF is con inuous wi h he enal idge (RR). The mesenchymal cells o all hese s uc u es belong o a WT1-exp essing cell lineage. (C)W 1 LacZ emb yo, E11.5. Ac i a ion o he W 1 epo e can be seen in he li e meso helium. PHMP, PPF and RR. The RRs appea a he le el o he s omach (STO). OE: oesophagus. (D)W 1 C e ;R26R YFP emb yo, E11.5. Cells om he WT1-exp essing cell lineage (g een) a e mo e abundan a Figu e 1 con inued on nex page Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 3 o 17 Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine like con inuum o med by he PHMP and he PPFs, a p ocess which is pa allel o he as g ow h o he li e lobes du ing hese s ages (Figu e 1B,C). As seen in he W 1 C e labeled cells, he PPFs con inue pos e io ly as he neph ic idges, he p o- geni o issue o he mesoneph os (Figu e 1B and C). A loose mesenchymal issue appea s in he PPF be ween he mo e compac a angemen o he cells in he PHMP and in he enal idges, bu he commissu al a ea o he PPF is always cons i u ed by compac mesenchyme simila o ha om he PHMP (a ow in Figu e 2C). All hese mesenchymal popula ions a e de i ed om a WT1- exp essing cell lineage, as shown by wo models, he W 1 C e ;R26R LacZ (Figu e 1B) and he W 1 C e ; R26R YFP (Figu e 1D). Exp ession o he W 1 gene is also e ealed in he PPF and PHMP mesen- chyme by he WT1-LacZ epo e and by immunohis ochemis y (Figu es 1C and 3A, espec i ely). Epi helial and mesenchymal cells o he PHMP and mos an e io PPFs de i e om a cell lineage in which Ga a4 exp ession is d i en by he G2 enhance . This is demons a ed by he exp ession o YFP in G2-Ga a4 C e ;R26R YFP emb yos, and suppo s he exis ence o a p ocess o cell mig a ion om PHMP o PPFs (Figu e 1E,F). Howe e , he neph ic idges and he mesoneph os de i ed om hem show no YFP+ cells in his model, indica ing ha he p ogeni o s o hese issues de i ed om a di - e en cell lineage. Thus, G2-Ga a4 lineage acing allows o es ablishing a well-de ined limi be ween wo la e al mesode mal e i o ies ha we will call he ein he G2 + and he G2 - domains (see igu e 3B,C). In e es ingly, he gonad and he ad enal s oma we e YFP+ (Figu e 1G), and his can be ela ed wi h he ea ly exp ession o GATA4 in he coelomic epi helium, which will co e he geni al bu no he enal idges (Figu e 1H). Inciden ally, he GATA4 posi i e cells om he cen al pa o he ST do no exp ess YFP in G2-Ga a4 C e ;R26R YFP emb yos (Figu e 1F), an obse a ion con- sis en wi h he lack o epo e ac i i y in he G2- Ga a4 LacZ emb yos (Figu e 1A). These da a e eal a signi ican he e ogenei y be ween cen al and la e al mesenchymal popula ions in he ST. A clea asymme y was obse ed in he dis ibu ion o PHMP mesenchyme in emb yos o E10.5- E12.5 (Figu e 1D,E). Mesenchymal cells we e always mo e abundan in he igh PHMP han in he le , which migh a o he he nia ion in he le side. We es ima ed, by image analysis, he olume aken up by he YFP+ cells in he igh and le PHMP o ou G2-Ga a4 C e ; R26R YFP emb yos. The R/ L a io o hese olumes was 1,21 and 1,45 in wo E11.5 emb yos, and 1,53 and 1,61 in wo E12,5 emb yos (mean = 1,45; S.E.M = 0,086). The - es o one sample compa ed wi h an expec ed alue = 1 (symme ical dis ibu ion) ga e a esul o 5,21 (p- alue=0,0069 o h ee d.o. ). Thus, he igh PHMP con ains abou 50% mo e cells om he G2-Ga a4 lineage han he le one. WT1 condi ional knockou mice display Bochdalek’s he nia This is a basically desc ip i e s udy o he pheno ype o mouse emb yos wi h condi ional dele ion o W 1 in la e al mesode m. We ha e included in he s udy 36 ou 104 mu an emb yos de ec ed by geno yping. The es o hem had been p e iously used o a s udy o he ca diac pheno ype (Cano e al., 2016). To unde s and he ole o WT1 posi i e cells in he de elopmen o diaph agm, we condi ionally inac i a ed W 1 gene using he desc ibed G2-Ga a4 C e line. Dele ion o WT1 in he PHMP/PPFs con- inuum, i.e. in he G2 + domain, causes a pheno ype cha ac e ized by de ec s in he in low ac o he hea , co ona y essels (Cano e al., 2016) and, impo an ly, Bochdalek’s he nia, mos equen ly Figu e 1 con inued he igh PHMP (R) as compa ed wi h he le one (L). No e he lack o YFP+ cells in he cen al a ea o he ST. AO: ao a; LU: lung. (E) G2-Ga a4 C e ; R26R YFP emb yo, E11.5. Cells om he lineage exp essing GATA4 unde he con ol o he G2 enhance a e s ained in g een, and RALDH2 is s ained in ed. Mos cells in he PHMP and PPFs belong o he G2-Ga a4 lineage, bu hey a e e y sca ce in he cen al a ea o he ST. Abundan YFP+ cells a e p esen do sally o he ao a, and a ound he no ocho d (NC). (F) G2-Ga a4 C e ;R26R YFP emb yo, E11.5. GATA4 immunos aining in ed. Colocaliza ion o GATA4 and YFP is obse ed in he PHMP and PPF, bu he cen al a ea o he ST shows GATA4 exp ession no d i en by he G2 enhance . (G) G2- Ga a4 C e ; R26R YFP emb yo, E11.5. Smoo h muscle alpha-ac in is s ained in ed. Gonads (GON) and ad enals (AD) con ain a la ge numbe o G2-Ga a4 lineage cells, bu hey a e sca ce in o he mesoneph os (MN). Da a eused, wi h pe mission, om Figu e 4A, Mun ˜oz-Cha ´puli e al. De elopmen al Dynamics, Special Issue: Mechanisms o Mo phogenesis, 245:307–322 (2016). Ó2015 Wiley Pe iodicals, Inc. (H) Immunolocaliza ion o GATA4 ( ed) in an E10.5 G2-Ga a4 C e ; R26R YFP emb yo. The G2 lineage is shown in g een. The e is a sha p bounda y (a ow) be ween he meso helial cells exp essing GATA4 o he geni al idge (GR), close o he do sal mesen e y (DM), and he meso helial cells o he enal idges (RR), which a e GATA4-. AO: ao a; OE: oesophagus. DOI: 10.7554/eLi e.16009.002 Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 4 o 17 Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine loca ed in he le side (Figu e 2). The geno ypes o he emb yos ob ained a di e en ages show he expec ed pe cen age o mu an s a all emb yonic s ages (abou 25%) excep o he s age E15.5, when we only ob ained ou mu an s among 60 emb yos (Table 1). The numbe o emb yos s udied in olde s ages (42 emb yos, nine mu an s, 21,4%), was no enough o con i m i he mu a- ion causes some le hali y in la e ges a ion. We analyzed WT1 mu an emb yos a di e en s ages o de elopmen . De ec in he PHMP in G2-Ga a4 C e ;W 1 l/ l mu an emb yos could be obse ed as ea ly as E10.5, since less mesenchymal cells a e p esen be ween he coelomic epi helium and he hepa oblas s when compa ed wi h con- ol li e ma es (Figu e 2A–D). A la e s ages, he closu e o he pleu al ca i y by g ow h o he ST/ PPFs c escen is delayed wi h espec o he con ols, and he mesenchymal cells o he PHMP a e Figu e 2. Pheno ype o he G2-Ga a4 C e ; W 1 l/ l emb yos. (A–D) Wild ype (A,C) and mu an (B,D), E10.5 li e ma es. The la ge amoun o mesenchymal cells in he pos hepa ic mesenchymal pla e (PHMP) is e iden in he wild ype, especially in he igh side. Se ial sec ions co esponding o he connec ion be ween he PHMP and he pleu ope i oneal olds (PPF) a e shown in C. No e he p esence o compac mesenchyme in he PHMP and also in he closes pa o he PPF (a ow in C). A co esponding sec ion o he mu an in shown in D. No e he lack o PHMP, he coelomic epi helium lying di ec ly on he hepa ic issue (a ows in D) and he limi o he sep um ans e sum (ST) mesenchymal cells (a owhead in D), which do no ex end la e ally. (E) Se ial sec ions o a G2-Ga a4 C e ; W 1 l/ l E11.5 emb yo a le els equi alen o hose shown in Figu e 1C. Despi e he p esence o no mal PHMP in he an e io pa , he pos e io a eas o he li e lack o la e al mesenchymal cells (a ow). The mesenchyme is es ic ed o he cen al ST. Renal idges (RR) appea a a le el co esponding o he en ance o he oesophagus (OE) in o he ST. MG: mesogas ium. (F) Wild ype E13.5 emb yo showing comple e isola ion o he pleu al ca i ies by he pleu ope i oneal memb anes ha cons i u e he main pa o he diaph agm (D). (G) G2- Ga a4 C e ; W 1 l/ l E14.5 emb yo a eigh di e en le els showing le diaph agma ic de ec wi h he nia ion o he le li e lobe (LI) in o he pleu al ca i y and se e e hypoplasia o he le lung (LL). Ec opic muscle appea in he medias inum (MM). Ad enals (AD), mesoneph os (MN), gonads (GON) and spleen (SP) appea no mal. LU: lungs; H: hea ; RL: igh lung; STO: s omach. DOI: 10.7554/eLi e.16009.003 Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 5 o 17 Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine loca ed mo e medially, no in he la e al ips o he li e lobes. This is mo e e iden a he le side (a ow in Figu e 2E). Thus, hese cells canno mig a e owa ds he PPFs p ecluding caudal g ow h o he la e al commissu es o he pleu ope i oneal sep a and closu e o he pleu al ca i ies. Fi e E12.5 mu an emb yos s udied al eady showed s ong educ ion o he PHMP size as com- pa ed wi h con ol li e ma es, bu he diaph agma ic de ec o he WT1 condi ional knockou emb yos became clea ly appa en by E13.5, when he pleu al ca i ies a e almos comple ely isola ed in wild ype emb yos (Figu e 2F). O 23 mu an emb yos analyzed by he s ages E13.5 o olde , only ou o hem showed no mal de elopmen o he diaph agm, while he es o emb yos showed di - e en deg ees o diaph agma ic de ec s (Table 1). Th ee o he emb yos showed hin, memb anous diaph agm in he le side and 16 showed a wide de ec in he diaph agm, equen ly wi h he nia ion o he li e in o he le pleu al ca i y and hypoplasia o he le lung (Figu e 2G). One o hem (E13.5) showed he de ec only a he igh side and he CDH was p esen in he le side in he es o hem. Thus, abou 80% o he E13.5 o olde mu an emb yos showed abno mal diaph agma ic de elopmen . Renal idges appea well de eloped in mu an E11.5 emb yos a a le el co esponding o he en ance o he oesophagus in o he ST (Figu e 2E). The p esence o in e media e mesode m in hese ho acic enal idges was con i med by ISH o Pax2 (Figu e 4). In wild ype emb yos o he same age ho acic enal idges can also be seen, bu loca ed mo e caudally, a he le el o he s om- ach (Figu e 1C). Figu e 3. WT1, GATA4 and RALDH2 exp ession in G2-Ga a4 C e ; W 1 l/ l emb yos. (A,B) Immunolocaliza ion o WT1 (A) and GATA4 (B) in wild ype E11.5 emb yos. The pos hepa ic mesenchymal pla e (PHMP) and pleu ope i oneal olds (PPF) show abundan posi i e cells. Howe e , he WT1 immuno eac i e cells a e p esen in he base o he PPF and he adjacen body wall, whe e GATA4+ cells a e absen . This di e ence de ines he G2+ and he G2- domains. Da a in A eused, wi h pe mission, om Figu e 3E, Mun ˜oz-Cha ´puli e al. De elopmen al Dynamics, Special Issue: Mechanisms o Mo phogenesis, 245:307–322 (2016). Ó2015 Wiley Pe iodicals, Inc. (C–D) Immunolocaliza ion o WT1 (C) and GATA4 (D) in G2-Ga a4 C e ; W 1 l/ l E11.5 emb yos. The PHMP and he G2+ domain o he PPF show GATA4+ cells bu no WT1+ cells due o he condi ional dele ion o his gene in he G2+ domain. WT1 exp ession emains in he G2- domain o he base o he PPF and body wall. GATA4 exp ession is no mal in mos cephalic PPF, bu immuno eac i i y disappea s in mo e caudal a eas o PPF, as shown in D’ and D’’. (E–H) Immunolocaliza ion o RALDH2 in E10.5 (E,F) and E11.5 (G,H) wild ype and G2-Ga a4 C e ; W 1 l/ l mu an emb yos. RALDH2 immuno eac i i y is high in PPF and he adjacen body walls. No e s ong immuno eac i i y in he enal idges (RR) o he E11.5 mu an emb yo. Howe e , RALDH2 immuno eac i i y is educed o absen in he PHMP and li e meso helium o he mu an emb yos, as compa ed wi h he con ols. CV: ca dinal eins; LU: lungs. DOI: 10.7554/eLi e.16009.004 Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 6 o 17 Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine The sha p bounda y be ween he abo e explained G2 + and G2  domains was clea ly obse ed by he immunolocaliza ion o WT1 and GATA4 p o ein in G2-Ga a4 C e ;W 1 l/ l mu an emb yos (Fig- u e 3). WT1 immuno eac i i y is s ong in he PHMP and PPF o he wild ype emb yos and localizes in he coelomic epi helium and, wi h a lowe in ensi y, in mesenchymal cells (Figu e 3A). Howe e , WT1 is clea ly down egula ed in he G2 + domain o he mu an emb yos indica ing an e icien Table 1. Numbe o emb yos s udied and % o G2-Ga a4 C e ;W 1 l/ l ound. The i e E12.5 emb yos showed abno mal pos hepa ic mesenchymal pla es a he le side, bu hey we e no compu ed o he diagnosis o he diaph agma ic he nia, ha was pe o med only on he 23 emb yos a he s ages E13.5 o olde . S age Numbe o emb yos s udied Numbe and% G2C e+; W 1 l/ l Emb yos analyzed o pheno ype No de ec Mild de ec Diaph agma ic he nia E10–10.5 42 11 (26,2%) 3 E11.5 89 21 (23,6%) 5 E12.5 76 21 (27,6%) 5 (5) E13,5 65 19 (27,1%) 13 1 1 11* E14,5 53 17 (32,1%) 4 2 2 E15,5 60 4 (6,7%) 4 2 2 E16,5 21 4 (19,0%) 1 1 E17,5– 18,5 21 5 (23,4%) 1 1 To al 427 102 (23,6%) 36 4 (17,4%) 3 (13,0%) 16 (69,6%) *We ha e included se en emb yos om emales ed wi h con ol die in he RA- escue expe imen . One emb yo showed CDH only a he igh side. DOI: 10.7554/eLi e.16009.005 Figu e 4. Localiza ion o Pax2 exp ession by in si u hyb idiza ion. (A) Con ol E11.5 emb yo. Pax2 is exp essed in he neu al ube (NT). The pleu ope i oneal olds (PPF) lack o Pax2 exp ession. (B) G2-Ga a4 C e ; W 1 l/ l E11.5 emb yo. Pax2 is exp essed in he PPF a he le el o he lung buds (LU). The exp ession is s onge in he ubula s uc u es ha a e de eloping in he le PPF (inse ). DOI: 10.7554/eLi e.16009.006 Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 7 o 17 Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine excision o he WT1 loxed allele (Figu e 3C). Immunolocaliza ion o GATA4 p o ein ma ked he bo - de be ween bo h G2 domains, con i ming he absence o endogenous GATA4 p o ein and he inac- i i y o he G2 enhance in he G2 - domain. (Figu e 3B,D). The p esence o GATA4+ cells in he mo e cephalic pa o he PPF suppo s a mig a ion o PHMP cells owa ds he PPFs. Since WT1 is in ol ed in epi helial-mesenchymal ansi ion (EMT) o he epica dium by ep ession o E-cadhe in and ac i a ion o Snail1 (Ma ı´nez-Es ada e al., 2010), we checked he exp ession o E-cadhe in in he de eloping PHMP o G2-Ga a4 C e ;W 1 l/ l mu an emb yos. Con ol E11.5 emb yos show an E-cadhe in nega i e coelomic epi helium o e a laye o mesenchymal cells in he de elop- ing PHMP (Figu e 5A–D). Howe e , mu an emb yos show exp ession o E-cadhe in in he epi he- lium o he co esponding a ea and lack o E-cadhe in nega i e mesenchymal cells (Figu e 5E–H). Sys emic WT1 loss o unc ion leads o comple e lack o PHMP We ha e compa ed he G2-Ga a4 C e ;W 1 l/ l CDH pheno ype wi h ha displayed by he sys emic WT1 knockou mouse emb yos, which do no su i e beyond E13.5. Despi e he ea ly emb yonic dea h o hese models, we ha e obse ed a mo e se e e de ec , wi h a comple e lack o PHMP and displaying, in some cases, pe sis en ho acic neph ic idges in E12.5 and E13.5 emb yos (Figu e 6). In hese emb yos only he cen al pa o he ST is conse ed, i.e. he a ea whe e he G2 enhance is no ac i e, as shown in Figu e 1E. Re inoic acid pa ially escues he de ec s in he diaph agm o WT1 condi ional knockou emb yos The e inoic acid (RA) signaling pa hway has been in ol ed in he o ma ion o diaph agm. To es whe he RA signaling migh be dis u bed in ou CDH model, we checked he exp ession o RALDH2 by immuno luo escence in G2-Ga a4 C e ; W 1 l/ l mu an and con ol emb yos. RALDH2 exp ession is ound in he PHMP and PPF o wild ype E10.5-E11.5 emb yos (Figu e 3E,G). The PPF o he G2- Figu e 5. Immunolocaliza ion o E-cadhe in in he le pos hepa ic mesenchymal pla e (PHMP) o ou E11.5 con ol emb yos (A–D) and ou E11.5 G2- Ga a4 C e ; W 1 l/ l emb yos (E–H). E-cadhe in appea s up egula ed in he coelomic epi helium o he PHMP o he mu an emb yos (a ows in E–H). In wild ype emb yos he coelomic epi helium o he PHMP lacks o E-cadhe in exp ession (a ows in A–D) and co e s a laye o E-cadhe in-nega i e mesenchymal cells (as e isks). DOI: 10.7554/eLi e.16009.007 Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 8 o 17 Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine Ga a4 C e ; W 1 l/ l mu an emb yos show an inc eased immuno eac i i y o RALDH2 bu we ound a educed o absen exp ession o his enzyme in he PHMP (Figu e 3F,H). This p obably e lec s, as discussed below, he pe sis ence o he in e media e mesode m o he neph ic idges in o he PPF. In ac , RALDH2 is no mally exp essed a high le els in he in e media e mesode m e en in sys emic WT1 knockou emb yos, which also show dec eased RALDH2 immuno eac i i y in he li e meso- helium (Figu e 6C,D). RA ea men pa ially escues he pulmona y pheno ype and p omo es al eologenesis in he ni o en model o induced CDH in a s (Mon edonico e al., 2008;Sugimo o e al., 2008). In o de o check he e ec s o a RA ea men on ou mu an pheno ype, in a i s expe imen we ed p eg- nan emales wi h a RA-supplemen ed die . Fo e hical easons we decided o limi he s udy o ou li e s in which we de ec ed eigh G2-Ga a4 C e ; W 1 l/ l mu an emb yos. Fi e o hem (62,5%) dis- played a comple e sep a ion o he pleu al ca i y by E15.5, i.e. abou h ee imes he p opo ion o no mal pheno ypes ound in he non- ea ed mu an s (Z es = 2,09, wo- ailed p- alue= 0,037). Con- ol li e ma es we e no a ec ed by he RA- ea men . Figu e 6. Diaph agma ic pheno ype in sys emic WT1 loss o unc ion. (A,B) WT1-/- E13.5 emb yos. The comple e lack o pos hepa ic mesenchymal pla e is e iden , mesenchymal cells a e absen om he la e al ends o he li e lobes and emain es ic ed o he cen al a ea o he sep um ans e sum (ST), close o he oesophagus (OE). The pleu al ca i ies a e con inuous wi h he pe i oneal ca i y. No e he enal idges (RR) pe sis ing a ho acic le el, and he abno mal adhesion o he igh li e lobe wi h he body wall (a ow in B). AO: ao a; LI: li e ; LU: lungs. (C,D) Immunolocaliza ion o RALDH2 in WT1-/- emb yos, E11.5 (A) and E12.5 (B). RALDH2 immuno eac i i y is s ong in he enal idges (RR), bu weak o absen in he li e (LI) meso helium (MES). AO: ao a; LU: lung. DOI: 10.7554/eLi e.16009.008 Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 9 o 17 Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine Delgado I, Ca asco M, Cano E, Ca mona R, Ga cı ´a-Ca bone o R, Ma ı ´n-Go ´ mez LM, So ia B, Ma ı ´n F, Cano DA, Mun ˜oz-Cha´puli R, Rojas A. 2014. GATA4 loss in he sep um ans e sum mesenchyme p omo es li e ib osis in mice. Hepa ology 59:2358–2370. doi: 10.1002/hep.27005 Dingemann J, Doi T, Gosemann J-H, Ru ens ock EM, Nakazawa N, Pu i P. 2013. Dec eased exp ession o GATA4 in he diaph agm o ni o en-induced congeni al diaph agma ic he nia. Bi h De ec s Resea ch Pa B: De elopmen al and Rep oduc i e Toxicology 98:139–143. doi: 10.1002/bd b.21037 Dingemann J, Doi T, Ru ens ock E, Pu i P. 2011. Exp ession o he Wilm’s umo gene WT1 du ing diaph agma ic de elopmen in he ni o en model o congeni al diaph agma ic he nia. Pedia ic Su ge y In e na ional 27:159–163. doi: 10.1007/s00383-010-2795-y D essle GR, Deu sch U, Chowdhu y K, No nes HO, G uss P. 1990. Pax2, a new mu ine pai ed-box-con aining gene and i s exp ession in he de eloping exc e o y sys em. De elopmen 109:787–795. Essa i A, Webb A, Be y RL, Sligh J, Bu n SF, Sp aggon L, Velecela V, Ma inez-Es ada OM, Wil shi e JH, Robe s SG, B owns ein D, Da ies JA, Has ie ND, Hohens ein P. 2011. A w 1-con olled ch oma in swi ching mechanism unde pins issue-speci ic wn 4 ac i a ion and ep ession. De elopmen al Cell 21:559–574. doi: 10. 1016/j.de cel.2011.07.014 Good ich ES. 1930. S udies on he S uc u e and De elopmen o Ve eb a es. London: Macmillan. Guadix JA, Ca mona R, Mun ˜oz-Cha´puli R, Pe´ ez-Poma es JM. 2006. In i o and in i o analysis o he asculogenic po en ial o a ian p oepica dial and epica dial cells. De elopmen al Dynamics 235:1014–1026. doi: 10.1002/d dy.20685 Hayashi S, Fukuzawa Y, Rod ı ´guez-Va ´zquez JF, Cho BH, Ve dugo-Lo´ pez S, Mu akami G, Nakano T. 2011. Pleu ope i oneal canal closu e and he e al ad enal gland. The Ana omical Reco d: Ad ances in In eg a i e Ana omy and E olu iona y Biology 294:633–644. doi: 10.1002/a .21351 Ijpenbe g A, Pe´ ez-Poma es JM, Guadix JA, Ca mona R, Po illo-Sa´nchez V, Macı ´as D, Hohens ein P, Miles CM, Has ie ND, Mun ˜oz-Cha´puli R. 2007. W 1 and e inoic acid signaling a e essen ial o s ella e cell de elopmen and li e mo phogenesis. De elopmen al Biology 312:157–170. doi: 10.1016/j.ydbio.2007.09.014 I i ani I. 1984. Expe imen al s udy on emb yogenesis o congeni al diaph agma ic he nia. Ana omy and Emb yology 169:133–139. doi: 10.1007/BF00303142 Klaassens M, Sco DA, an Doo en M, Hochs enbach R, Eussen HJ, Cai WW, Galjaa d RJ, Wou e s C, Poo M, Laudy J, Lee B, Tibboel D, de Klein A. 2006. Congeni al diaph agma ic he nia associa ed wi h duplica ion o 11q23-q e . Ame ican Jou nal o Medical Gene ics Pa A 140:1580–1586. doi: 10.1002/ajmg.a.31321 K eidbe g JA, Sa iola H, Lo ing JM, Maeda M, Pelle ie J, Housman D, Jaenisch R. 1993. WT-1 is equi ed o ea ly kidney de elopmen . Cell 74:679–691. doi: 10.1016/0092-8674(93)90515-R Ku pios NA, Iban ˜es M, Da is NM, Lui W, Ka z T, Ma in JF, Izpisu ´a Belmon e JC, Tabin CJ. 2008. The di ec ion o gu looping is es ablished by changes in he ex acellula ma ix and in cell:cell adhesion. PNAS 105:8499– 8506. doi: 10.1073/pnas.0803578105 Ma ı´nez-Es ada OM, Le ice LA, Essa i A, Guadix JA, Sligh J, Velecela V, Hall E, Reichmann J, De enney PS, Hohens ein P, Hosen N, Hill RE, Mun ˜oz-Chapuli R, Has ie ND. 2010. W 1 is equi ed o ca dio ascula p ogeni o cell o ma ion h ough ansc ip ional con ol o Snail and E-cadhe in. Na u e Gene ics 42:89–93. doi: 10.1038/ng.494 Maye S, Me zge R, Klu h D. 2011. The emb yology o he diaph agm. Semina s in Pedia ic Su ge y 20:161– 169. doi: 10.1053/j.sempedsu g.2011.03.006 Me ell AJ, Ellis BJ, Fox ZD, Lawson JA, Weiss JA, Ka don G. 2015. Muscle connec i e issue con ols de elopmen o he diaph agm and is a sou ce o congeni al diaph agma ic he nias. Na u e Gene ics 47:496– 504. doi: 10.1038/ng.3250 Mon edonico S, Nakazawa N, Pu i P. 2008. Congeni al diaph agma ic he nia and e inoids: sea ching o an e iology. Pedia ic Su ge y In e na ional 24:755–761. doi: 10.1007/s00383-008-2140-x Moo e AW, Schedl A, McInnes L, Doyle M, Heckshe -So ensen J, Has ie ND. 1998. YAC ansgenic analysis e eals Wilms’ umou 1 gene ac i i y in he p oli e a ing coelomic epi helium, de eloping diaph agm and limb. Mechanisms o De elopmen 79:169–184. doi: 10.1016/S0925-4773(98)00188-9 No den J, G ieskamp T, Ch is o els VM, Moo man AF, Kispe A. 2012. Pa ial absence o pleu ope ica dial memb anes in Tbx18- and W 1-de icien mice. PLoS One 7:e45100. doi: 10.1371/jou nal.pone.0045100 No den J, G ieskamp T, Lausch E, an Wijk B, an den Ho MJ, Engle C, Pe y M, Momme s eeg MT, Ch is o els VM, Niede ei he K, Kispe A. 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 cula ion Resea ch 106:1212–1220. doi: 10.1161/CIRCRESAHA.110.217455 Pa is ND, Coles GL, Acke man KG. 2015. W 1 and b-ca enin coope a i ely egula e diaph agm de elopmen in he mouse. De elopmen al Biology 407:40–56. doi: 10.1016/j.ydbio.2015.08.009 Pobe BR, Russell MK, Acke man KG. 2010. Congeni al diaph agma ic he nia o e iew. In: Pagon R. A, Adam M. P, A dinge H. H, Wallace S. E, Amemiya A, Bean L. J. H, Bi d T. D, Fong C. T, Me o d H. C, Smi h R. J. H, S ephens K (Eds). GeneRe iewsÒ[In e ne ]. Uni e si y o Washing on: Sea le. p 1993–2015. P ege -Ben Noon E, Ba ak H, Gu mann-Ra i N, Reshe R. 2009. In e play be ween ac i in and Hox genes de e mines he o ma ion o he kidney mo phogene ic ield. De elopmen 136:1995–2004. doi: 10.1242/de . 035592 Rojas A, De Val S, Heid AB, Xu SM, B is ow J, Black BL. 2005. Ga a4 exp ession in la e al mesode m is downs eam o BMP4 and is ac i a ed di ec ly by Fo khead and GATA ansc ip ion ac o s h ough a dis al enhance elemen . De elopmen 132:3405–3417. doi: 10.1242/de .01913 Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 16 o 17 Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine Sco DA, Coope ML, S ankiewicz P, Pa el A, Po ocki L, Cheung SW. 2005. Congeni al diaph agma ic he nia in WAGR synd ome. Ame ican Jou nal o Medical Gene ics Pa A 134:430–433. doi: 10.1002/ajmg.a.30654 Sugimo o K, Takayasu H, Nakazawa N, Mon edonico S, Pu i P. 2008. P ena al ea men wi h e inoic acid accele a es ype 1 al eola cell p oli e a ion o he hypoplas ic lung in he ni o en model o congeni al diaph agma ic he nia. Jou nal o Pedia ic Su ge y 43:367–372. doi: 10.1016/j.jpedsu g.2007.10.050 Su i M, Kelehan P, O’neill D, Vadeya S, G an J, Ahmed SF, Tolmie J, McCann E, Lam W, Smi h S, Fi zpa ick D, Has ie ND, Rea don W. 2007. WT1 mu a ions in Meacham synd ome sugges a coelomic meso helial o igin o he ca diac and diaph agma ic mal o ma ions. Ame ican Jou nal o Medical Gene ics Pa A 143A:2312–2332. doi: 10.1002/ajmg.a.31924 Volpe MV, Wang KTW, Nielsen HC, Chinoy MR. 2008. Unique spa ial and cellula exp ession pa e ns o Hoxa5, Hoxb4, and Hoxb6 p o eins in no mal de eloping mu ine lung a e modi ied in pulmona y hypoplasia. Bi h De ec s Resea ch Pa A 82:571–584. doi: 10.1002/bd a.20481 on Gise A, Zhou B, Hono LB, Ma Q, Pe yk A, Pu WT. 2011. WT1 egula es epica dial epi helial o mesenchymal ansi ion h ough b-ca enin and e inoic acid signaling pa hways. De elopmen al Biology 356: 421–431. doi: 10.1016/j.ydbio.2011.05.668 Wessels A, an den Ho MJ, Adamo RF, Phelps AL, Lockha MM, Sauls K, B iggs LE, No is RA, an Wijk B, Pe ez-Poma es JM, De man RW, Bu ch JB. 2012. Epica dially de i ed ib oblas s p e e en ially con ibu e o he pa ie al lea le s o he a io en icula al es in he mu ine hea . De elopmen al Biology 366:111–124. doi: 10.1016/j.ydbio.2012.04.020 Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 17 o 17 Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine