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between thisversion andtheVersion ofrecord. Please citethisarticle asdoi:10.1002/ajmg.c.31612. through thecopyediting, typesetting, pagination andproofreading process,whichmay lead todifferences This istheauthor manuscriptaccepted forpublicationandhas undergonefullpeerreview buthasnotbeen KEYWORDS: primarilytoalteredduesignaling pathway.viaShh the Nodal, TGIF2, SHH appears appears thethat defectspatterningforebrain and HPEin developmentalareprocessesthat regulatedby TGIFs Nodal-dependent,bemay it pathwaySonicHedgehogand (SHH) independently.signaling someAlthough suggest that regulateTGIFs the TransformingGrowth (TGF)Factorß/Nodal signaling light on howlight human proteins and ofproteins analysis of analysisdevelopmentforebrainin of mouseembryos both lacking models basedlossmodels on of of function contributegene tothepathogenesis ofHPEhas remainedelusive. However,mouse frequently HPEinscreened patients,genes understanding ofan howinmutations this TGIF1 and humans, potentialcausative ofat14 genes loci thesehave identified. been Although andboth genetic environmental Multiplecauses. haveassociated been withloci HPEin Holoprosencephalyais (HPE) human frequent developmentalforebrain with disorder mammaliandevelopment. MedicalHismajorSchool.interest understanding in is associatedmechanisms with early KenichiroTaniguchi,ResearchPh.D.is atInvestigatorUniversity Michiganthe of expressionbetaby TGF earlyduringsignaling cancerdevelopmentand progression. VirginiaUniversity Medical of HisSchool.onin themain regulation focus of gene DavidaPh.D.is Professor of Biochemistry Molecularand Wotton, attheGenetics DAVIDWOTTON DevelopmentBrainNormal andHoloprosencephaly. FunctionsTGIFofHomeodomain Proteins andRolesTheir in (originally TGIF, for Thymine Factor)Guanine-Interacting amongis the most

Center CellCenter for Signaling, University Virginia,Charlottesville, of USA Author Manuscript

holoprosencephaly, development, forebrain, mouse, TGIF, TGIF1, 1 TGIF1 DepartmentofBiochemistry Molecularand Genetics,and 1 TGIF1 * * This article isprotected by copyright. All rights reserved. singlenucleotide variants fromHPEpatients, combinedwith AND variants might cause FunctionalanalysesHPE. of TGIF KENICHIRO TANIGUCHI TANIGUCHI KENICHIRO Tgif1

Wiley-Phase 1 ,and therelated 2 Tgif

Tgif2 mutant mouseembryos is gene,havesome shed Tgif1 and Tgif2 ,

Author Manuscript Cell Center for Signaling, *Correspondence:

University of MichiganUniversity Medical Arbor.AnnSchool,of USA 2 DepartmentofCell Developmental and Biology, This article isprotected by copyright. All rights reserved. a: 434-924-1236 fax: 434-243-6752 voice: [email protected] CharlottesvilleVA22908 BoxHSC, 800577, Virginia,University of John Wiley&Sons Wiley-Phase 1

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rat CRBPII rat ( Human wasidentifiedTGIF1 by its first ability a specificto bindto DNA element the from HOMEODOMAINTGIF PROTEINS HPE. causes pathways interrogate the regulatedandby Tgifs howunderstandto ofloss functionTgif links TGIF2, their andtoHPE, analyses of mouse havemutants that been generatedto patients (Solomonetal., we Here 1993).discuss the analysisfunctional and of TGIF1 commonlyare most screened as mutation partfor ofroutine evaluationgenetic ofHPE superfamilycharacterizedis Amino byThree acid a Extension Loop betweenpresent the importantnumerousdevelopmental functions(T.Affolter,Burglin & R. 2016). TALE The homeodomainisin found plants,animals, and fungi homeodomainand proteins regulate DNAinteractions. Originallyidentifiedtheinhomeotic ingenes primarily positions, withinthethird carboxyl-terminal helix,sequence-specific mediate Affolter, Burglin,(Gehring, & 1994; J. GehringSpecificetal.,1994). aminoW. acid proteinbindingand domain,consisting helicesofthree athat structureform globular Burglin,Mukherjee& 2007). homeodomain approximatelyanThe is amino60 DNAacid superfamilyhomeodomain of proteins 1995; al., (Bertolino et Burglin,T.R. 1997; &Melhuish, Gallo, 2001). and TGIF1 areWotton, membersTGIF2of atypical the TALE highlyconservedhomeodomains proteins the sharelimited similarityetal.,2000; (Imoto been associatedbeen with HPE,non-syndromic and the phenotypes(Solomon, &Gropman, Muenke, 1993).least14 At candidate havegenes isthere and considerablevariability both inthe severity penetrance the oftheand neurologicalincludingHPE, defects,hydrocephalussuch as orcognitive impairment, Beachy,Inaddition,1998). aof othernumber abnormalitiescan be associated with whichassociatedis with defectivemidlinestructures 1998;facial (Golden, Rubenstein& defectprimary HPEinisof a theventralfailure todivideforebrain twohemispheres,into the human most is frequent developmental forebrain disorder (Leonciniet2008). al., The HPEaffectsapproximately liveupand births1/8000 to1/250 conceptuses,human and INTRODUCTION related related Reimund, Clerc,&1995; Bertolino, Wildt-Perinic, Richards, Wildt, Clerc,& 1996). The

AuthorTGIF2 Manuscript

Rbp2)and promoter, mousegene wasTgif1 clonedby homology(Bertolino, was identifiedalsobasedsimilarity on to This article isprotected by copyright. All rights reserved. John Wiley&Sons Wiley-Phase 1 SHH TGIF1 , , ZIC2 ,although outsidethe , , D.melanogaster SIX3 and TGIF1 , , the genes

Although haveconservedTGIFs a it notTALE,is loop inif known this the proteinsTgif Shen, Mann, Aggarwal,Shen, & 1999;Piper, Batchelor, Cleary, & Chang, 1999). Wolberger, homeodomainotherproteins, asthe incase of PBX-HOX interactions (Passner,Ryoo, DNAbinding, affect butlikely playsinteractioninrole a with proteins,other including twohelices first (T.R. Burglin, 1997;R. Burglin T. 2016).Affolter,& notTALEdoesThe SHH the14 Of candidatehave that beengenes associatedwith non-syndromic theHPE, VARIATIONTGIF1 HPE IN development(discussed below). transcriptionalrepressors appearand have largelyto overlapping functionsearlyin terminalrepressiondomain 1A), (Fig. thevertebrate and TGIF1paralogs TGIF2 areboth sequenceDespite differences outside thehomeodomain and conservedcarboxyl transcriptionalactivators rather than transcriptionalrepressorset(Hymanal.,2003). terminaltobutit,theyshareno similarity other vertebrate to the andTGIFs are thehighlywith conserved homeodomainplus aminothesequenceacid20 carboxyl Spagnoli Brivanlou,1995; & 2008). Incontrast, inare thereafliesof pair relatedproteins transcriptionalrepressors et(Hymanal., 2003;Ryan, May,Tejada, Dubaova,Deeley, & and havefish chicken been characterized,all having broadly similar functions as vertebrates. Inadditiontomouseand human, proteinsTgif-related zebrafrom Xenopus, Patterson,&(Hamid, Brandt,Close 2008). TGIF1homologs areand TGIF2 inpresent allsplicevariantsencode thehomeodomain and sequences carboxyl-terminal the toit isoformidentified, the major encodes aminothe272 proteinacidoriginally andidentified, form form proteins.the TGIF2 multiple spliceofvariantshuman While Boyd,& Lim, Chinnadurai, is1998), in present vertebrate orthologs, TGIF1 absentbutis familyof transcriptionalcorpressors (Chinnadurai,2002,2007; Schaeper, Subramanian, less similar.much A conservedsequence motif (PLDLS), toknownrecruit theCtBP whichproteins, interacts corepressors, with with oftheremainder thesequence being a isthere second highsequenceregion similarityof towardthecarboxyl-termini of the superfamily TALE Bartholin,Newfeld, (Hyman, & 2003)Wotton,(Fig. 1A). this,Outside carboxyl-terminaltothe homeodomainisthat nototherinconserved membersof the identitysequencewithin homeodomain, the and aminoa acid20 immediatelyregion protein-proteinmediatesspecific interactions. and haveTGIF1 highTGIF2 a degreeof , , ZIC2

, Author Manuscript SIX3

As withAs HPEothermutations,loss of dominant dominant manner. critical region atregion critical theHPE4at 18p11.3locus (Grippetal.,2000; Overhauseretal.,1995). no potentiallyno variantsinpathogenic the (Roessler & (Roessler Muenke, 2010). HPEresponsiblewhen eitherfor disrupted, geneticallyby environmentalteratogensor HPE major humans,ingene and thispathwayis the bestis characterized that being as additionalandcraniofacialdefects neural compared topatientswith intragenic ofclinical range butphenotypes, completedeletions oftheHPE4 locus maycause and in the human the in HPEphenotype. the However, asisthere yetno HPE-associatedevidence for mutations human human candidate that,whengene mutated, mightcooperate with a browser(GenomeAggregation Database; these19,affect Of ten aminoat whichacids arevariantsnotpresent in the gnomAD identifiedbeen1B) (Fig. etal.,2007;(El-Jaick etal.,2010; Keaton Mercier etal.,2011). suggeststhat Taniguchi,Anderson,Sutherland, & 2012), Wotton, embryonicdevelopment (Melhuish,& Taniguchi, 2016;PowersWotton, etal.,2010; Muenke,& 2012). Zhou, subtlemore differences genetic inresultsappearance of HPE the (Roessler,Velez, likely appears predisposinga that variant combinedwith environmentalotherand factors phenotype.needed the Althoughfor thispossibility notbeen has fullyitexcluded,also suggestsmutations a two hit model,where atvariants twocommonlylociare affected somebeen has speculation incompletethat penetrance the ofHPE-associated to 30-40% to have variants the in HPEpatients (Solomon al.,1993). et Amongindividuals with historyHPE,a family upof 2016). nine remaining The variantsarealso foundthein includingdatabase,gnomAD relativelylowfrequency of variantspossiblerare that mighthave been missedthisinanalysis, particularly giventhe this locus this togetherwith variants(Keatonetal.,2010). perhapssuggests deletionThis ofthat additionalatgenes SIX3 Since Tgif1 andSinceTgif1in appear havemice Tgif2 largelyto overlapping functionsduring TGIF1 Author Manuscriptand TGIF2 havethat beenHPEin patients, singlefound 19amino-acid havechanges TGIF2

TGIF1

TGIF1 gene. Screening gene. ofa almost cohort HPEof 500patients revealed is unlikelyis have a to major role itHPEin humans, in remains variantsareeach in1-2% the range. Thus, This article isprotected by copyright. All rights reserved. TGIF1 variantsHPEin are associatedfound patients thewith full TGIF1 TGIF1 can contribute toa broaderrangeofphenotypes. There SHH variantsin additionHPE. In tothelarger indeletions was identified as present the gene in the minimal gene, John Wiley&Sons TGIF1 Wiley-Phase 1 TGIF2 http://gnomad.broadinstitute.org(Leketal., ) ZIC2 appearsbe inheritedto anin autosomal (El-Jaickgene etal.,2007). Although this variantsare foundonlyin5%, around TGIF2 representsa reasonable TGIF1 SHH mutationdrivingin appearsbe theto TGIF1

data thedata major272for amino-acid encoding isoformof are1C). thereno variants truncating (frameshiftandWhile nonsense)in the gnomAD halfcontrast, ofthemissense variants HPE are in from patients thehomeodomain (Fig. 23%withofmissense variantsin thehomeodomain ofthecodingsequence).(27% In represents 23%of represents thecodingsequence 1C). proportions(Fig. in The over the overthe population.wider Thevariants missense arerelativelyfrom gnomAD evenly distributed HPE,respect with althoughto some non-deleteriousvariantsbemay inpresent the activetranscriptionalrepressorslimit expression.to ofInsupportgene whenthis, TGIFs 2006).Wotton, itThusis in likelyadditioncompetingthat with activators,to recruit TGIFs thatthe motif is CtBP toknownrecruit corepressors (Melhuishal.,2001;Melhuish et & 1999).Massague, interactsalsoTGIF2 with histone deacetylasesand mSin3,butlacks 2001;Massague, & Lo,Lee,& Massague, 1999;Wotton, Lo,Swaby, & Wotton, (Melhuish2000;Sharma& Sun,&2001; Wotton, Knoepfler,Laherty,Wotton, Eisenman, transcriptionalcoreprerssors including histone deacetylases, mSin3, and CtBP1 1995). Subsequental., workhas shown that interacts TGIF1with multiple general drivenelementbyby this competing bindingRXRfor nuclearwith receptors (Bertolino et element, andresponse itwas suggestedthat limitcould TGIF1 transcriptional activity homologs. mouse initialThe ofidentification wasTGIF1 abilitybybind its to toa retinoid sequences,acidamino itlikelyis conclusions that theseanalyses from apply the to someanalysiswith of TGIF2. Givenhuman that mouseand sharealmost Tgifs identical ofmajorityThe functionalanalysis been has performedwithof TGIFs human TGIF1, TRANSCRIPTIONALREGULATIONBY TGIFS HPEpathogenesis. itpatients,perhaps suggests disruptionthat of thehomeodomain bemay important for variantsliewithin coding sequence basedthe is a relativelyon numberlimited HPE from function (discussedbelow). theAlthoughanalysis whereof missense and truncating closea frame-shift tothecarboxyl-terminus, and shownbeen has affect to TGIF1 variantsHPEpatients are within from the homeodomain. remainingThe variant inresults unaffected individuals. unaffected withconsistentisThis the lowofpenetrance significant proportionsignificant ofthe twohavethat shownbeen tohave consequencesfunctional(discussed below). Thus,a

Author ManuscriptTGIF1

coding sequence,with 18%inhomeodomain,ofthem the which This article isprotected by copyright. All rights reserved. TGIF1 variants found HPEinpatientsarein present John Wiley&Sons Wiley-Phase 1 TGIF1 , offour five truncating TGIF1 TGIF2 mutations aresimilar, Page 6of31 Page 7of31

TGIF1 competedTGIF1 with RXR bindingfor totheretinoid responseelementof the transcriptionalactivators etal.,2015;Yang(Willer etal.,2000). Meis the of TALE family homeodomainproteins, whichbindthe samesequence butare Inaddition,2012). been has TGIF1suggested direct to compete bindingfor toDNAwith Anderson etal.,2017;E. etal.,2015;Lee Zerlanko,Bartholin, Melhuish, & Wotton, independent levelsare of andthe TGFß/Nodal nuclearreceptor signaling pathways (A. reducedwith whichin levels, TGIF the ofmajority that genesexpressionin change 2A). (Fig. appears This consistenttobe withanalyses transcriptome cellsin or tissues cognate its independentresponseelement, ofother recruiting proteins(Leeet al.,2015) EScellssuggests themajorwaythat iswhich DNArecruitedis inTgif1 to via to binding Swaby, al.,1999).Lo, et wide Recent genome analysis ChIP-seq for by inTgif1 mouse drivetranscriptionalrepression linkedofa reporter (Melhuishgene al., et 2001; Wotton, artificially are toDNAby targeted a heterologous to DNA bindingfusion domain they transcription factorsare andphosphorylated, inaccumulate thenucleus, wheretheybind signalingTGFß/Nodal pathway. response In to TGFß familysignals, the SMAD Melhuishal.,2016). et butnot acid, increasedfor HPE-like phenotypesspecifically al.,(Bartholin et 2006; for increasedfor sensitivity of responsiveretinoid expressionbeengene has interest.of some someevidenceis There Chernoff,1995),Rogers,& thepossibility levelsreduced that resultinTGIF increased acidsretinoic linkand their toHPE-like phenotypes (Lanoue etal.,1997;Dehart, Sulik, need without a the consensusfor TGIF site(Fig. Given1B). theteratogenic effectsof possibility raises the that mightregulate additionalTGIFs NR regulated responses Mangelsdorf,2014), throughrecruitment interaction commonwith RXRthe partner nuclearothermultiple receptorsadditionin toretinoicreceptorsacid (RARs) (Evans& expressionprograms(Mangelsdorf et al.,1995).is common SinceRXR a partner for dimerizebindtoDNAand responseintoligand binding, to controlmany complexgene receptorsnuclear comprisea large(NR) transcriptional familyof regulators,which site consensus (Bartholinetal.,2006;Melhuish, Bjerke,& Chung, 2010). The Wotton, canonicalmorenuclear receptorbinding elements(NRE)without the need a for TGIF interactcan withTGIF1 such RXR,that couldTGIF1 recruitedbe indirectly DNAat to (Bertolinogene etal.,1995). However, analyses recent morehave suggested that

This competitionThis bindingfor toDNAconsistentis theoriginalwith suggestion that

Author inMuchtheinterest of hasTGIF centeredfunction on of the roles inTGIFs the Manuscript This article isprotected by copyright. All rights reserved. Tgif1 nullmouse embryos to John Wiley&Sons Wiley-Phase 1 in uteroin exposure to retinoic Rbp2

ubiquitylationdegradation,and orpreventing SMAD2 phosphorylation responseinto indirect mechanisms withinterfering for responses, TGFß as promotingsuch SMAD2 potentialofothernumber have functions for TGIF1 examined.been includeThese more additionalmechanism for specificity of theinTGIFpathway. function TGFß role bindinga for hasTGIF-DNA notbeen definitively andprovide out, ruled this might a recruitment viawithmodel SMADsinteraction However,1C). (Fig. it notedshould be that nottarget isgenes thoughtDNA binding to require byTGIFs,suggesting indirectan of subset TGFßresponses aresubject to regulation byTGIFs.Regulation SMAD of expressionrepresentsa ofoverall fraction TGIF and function, itpossibleis onlythat a of the TGFß pathway,signaling likelyisregulation that it of TGFß-responsive gene byboundalso (LeeetAlthoughTgif1 al.,2015). thiswithconsistentis as regulatorsTgifs orSmad3 Smad2 suggested thethat ofmajoritywere that boundgenes by were Smads comparisonofand Tgif1 genome-wideESin binding cells mouse with bybound regions activationforof are that genes TGFß responsivetheseincells(Zerlanko etal.,2012), Inprimary2012). mouse embryo fibroblastslackingwasthere someTgif1, enrichment withconsistentrole a for in pathwayTGIFs this al.,2010; et al.,Taniguchi (Powers et byofcaused loss inTGIFcan linked function bemice Nodal geneticallyto signaling, inducibleinmajor role or feedback ofmechanisms repression. which degree to cellsrespond transcriptionally signaling, but TGFß notplay do to a regulatessignaling directly.ledTGIFs hasThis thetomodel that levelsTGIF thelimit Stroschein, Zhou,Zhou,Luo, 1999),& Wang, therebeen has evidencelittle that TGFß of the TGFß pathway,signaling SMAD7 as SKILsuch (SnoN)or (Nakao etal.,1997; expression2001;(Melhuish al., Lo,Lee,et al.,1999). et Unlikeinhibitors otherWotton, of TGIF-bound transcriptionalandcorepressors, reduced responsive TGFß gene orTGIF1 withTGIF2 SMADs competitioninresults with SMAD recruitmentcoactivators, byitsbinding consensus siteto TGIF1 (Wotton, Lo,Lee,etal., 1999).of Interaction withinteract SMAD2 SMAD3, appearsbe this and and independent to ofdirectDNA expressionactivate gene (Hill,2016;Massague et 2005).al., was TGIF1 to found the(SBE),SMAD complexprimarily functionsto recruit transcriptionalcoactivatorsand conjunctioninwith thesharedpartner, SMAD4.bound aOnce SMAD to element binding Signaling Nodalfrom ActivinsTGFßs, and primarily is by SMAD2mediated SMAD3, and DNAand to regulate expressiongene (Hill,2016; Seoane, Massague, & 2005). Wotton,

Authoraddition In toregulation expressionof gene via recruitment totarget a genes, Manuscriptembryogenesis, During itappearsat leastthat a of subsetphenotypes the This article isprotected by copyright. All rights reserved. John Wiley&Sons Wiley-Phase 1 Page 8of31 Page 9of31

Although theAlthoughmajority changesof HPE-associated in FUNCTIONALANALYSIS VARIANTSOF TGIF1 transcription factors(Fig.2). corepressorsviatoDNA,combination a ofdirect interactionandDNA binding with other characterizedrepressorsof as expressiongene that recruit transcriptionalgeneral transcriptionalregulation (Melhuish 2000).& arebestThus, TGIFs Wotton, corepressorsCtBP (describedbelow), consistent HPEeffectsbeingwith due to on intragenic pathways as clue to the when arethat, disrupted, HPE.responsible Of the for 19 ofintragenicconsequences variantsareofsignificant interest as they may some give expressionetal.,(El-Jaick Although2007). atvariantsR91 the positionhave yet not interaction reduces with SMAD3alsoand RXR, and reduces repressionofreporter gene inDNApresentis binding the helix, abolishesbinding to the consensus site,TGIF homeodomain the haveshown been to reduce R90CTGIF1 function. whichThe variant, (El-JaickTGIF1 etal.,2007;Melhuish & 2000).variantswithin missense TwoWotton, showntodisruptinteractionwith both andCtBP1 reduce to transcriptional repressionby conserved motif (PLDLS) recruitsthatcorpressors, CtBP and variant this has been shownhave to consequences. functional S28CThe presentisvariant withinthe HPEpatients, from half have intested been functional butonlyassays, havethree been possiblydecreased to duestability (El-Jaick 2007). etal.,the 14 Of missensevariants protein, of atthe amino acidwas 260, shown verytoinresult low levelsof expression, thirdalpha-helixbinding 1C). (Fig. A close frame-shift mutation tothecarboxyl-terminus likelyis havefourth ato effectsincesimilarwould it at leastremove part oftheDNA- tested been functionallyinactive are (El-Jaick2007; etMar Hoodless, & al., 2006). The homeodomainNot(Fig.1).surprisingly, thethree these of truncation havemutants that Mercier etal.,2011),2010; in result truncationfour oftheprotein within the at leastone at variantin transcriptionaleffects. aretheThesemost extensively characterized TGIF and functions, expressionprogramsby mechanisms, multiplein review onthis we directthemore focus AXIN1by sequestering and AXIN2et al., (Zhang 2015). regulatemayTGIF1 While gene has TGIF1 recentlywherebyproposed,been TGIF1 indirectly activates signaling WNT et al.,2006;TGFß (Seo et Anal.,2004).Seo additional, non-transcriptional function for

Author ManuscriptTGIF1

variants HPEin patients found (El-Jaicketal.,2007;Keaton etal., This article isprotected by copyright. All rights reserved. TGIF1 from an HPEfrom patient specifically disruptsinteractionwith John Wiley&Sons Wiley-Phase 1 TGIF1 aredeletions, the functional

database, consistentdatabase, with theincomplete penetrance of variants haveof the that shownaffectbeen to arepresentalso function in the gnomAD of some thesevariants representchangessilent theinpopulation.present someIndeed, typeTGIF1,appropriateorthat the assaywas notused.However, alsois it possible that it assays,remains possible thisduethatis eitherpresence to the ofendogenous wild Given the lowGiven the andpenetrance relativelylowfrequency of FUNCTION LOSS MOUSEOF MODELS notprovidetheydo any to thecluespathwayssignaling affected. RXRSMADinteraction, or ordirectDNA binding independent of thesepathways, so ofthenonereduced functionvariantsdistinguish ofbetween repression transcription via ratherHPE,thannon-transcriptional other havethat been However,proposed.functions itclearlylossis suggests ofthe transcriptional repressionby contributes that TGIF1 to namelyfunction, corepressorreducing recruitment (Melhuish2000).& This Wotton, perhapsvariantisthe informative, most since athishas very effectonspecific protein variants missense shown tohave differences functionalfrom thewild S28C the type, important ifimportant to test to restore normal to restore proliferationin fibroblastslackingendogenous whereas Tgif1, theandS28C Q107Lvariantswere able variantwere unablealsocomplement ato proliferation defect primaryin mouseembryo P63AHPE-associated variant etal.,(El-Jaick 2007).P63R mutantThe and R90C not foundisHPEin patients,appears it tohave a effectonsimilar proteinto function the etal.,2007;JaickFerrand, Demange, Prunier,Seo,Atfi,& 2007). Although this change proteininaggregationresulting wasthat suggested tosequester wild(El- TGIF1 type P63Ralterationinhas beenalsoTGIF1, shown tohave consequences,functional SMAD3and interaction, reduces transcriptional repression etal.,2007).(El-Jaick A Jia,May, (Tejada, Deeley, &P63A variantThe 1999). decreasesalso DNAandbinding residuecontact that,when alteredtomethionine prevents consensus sitebinding tested,itbeenlikely is that theywillDNAbinding, since ais affect directDNA this of mice with of mice (Bartholin etal., (Bartholin 2006; McKinney,Jin, Gu, 2006;Ding,Mar& Hoodless, & Shen 2006; & isoform wasof the major removed,and none revealedanyphenotypes HPE-like

Author Manuscriptvariants didnotFor that resultapparentin defectsin functioncellinTGIF1 based Tgif1

deletions were created,whichin themajority of the codingsequence TGIF1 This article isprotected by copyright. All rights reserved. is indeedisan HPEcausal ingene mouse models. Severallines Tgif1 John Wiley&Sons null Hoodless,fibroblasts (Mar& 2006). Wiley-Phase 1 TGIF1 TGIF1 mutations. the Of variantsin wasHPE it Page 10of31 Page 11of31

and placentaldefectsand have observedbeenin a relativelyspecific. In C57BL6strain pure background, reducedviability, growthdelay variablewere generallyof andpenetrance often appearedbe backgroundto strain- 2005). numberWalsh, A defects wereof observed thesein mouse models,but they defects Tgifs in Tgifs Nodalregulating retinoidand signaling.Combining 2008).evidenceother Thus, model organismsisfrom broadly supportivea role of for synthesisacidand and responses, affect to forebrain(Gongalpatterning& Waskiewicz, fibroblasts from associatedasymmetry, alteredwithNodal signaling (Powersetal.,2010). Primary demonstration later that lacking bothembryos and Tgif1 showedTgif2 ofleft-rightloss 2006).Hoodless, identificationThe oflaterality defectsin supported this is model by the althoughmice, thesephenotypes didnotappear pure C57BL6 backgrounda in (Mar& & Houston,2008).& Inzebrafish, reducing 2006; Zerlanko 2006; etal.,2012). Increased sensitivityof background laterality background anddefects some growthdelay wereobservedthein alterationsinsignaling TGFß (Tateossianet al., Ina 2013). mixed129Sv/CD1 mediahave withotitis effusion(OME),resulting deafness, in potentially to linked etal.,2013).Tateossian Additionally,inTgif1background this mutants were shown to in this caseby this in controlling expression ofthe EvidenceXenopus a from supports inrole Nodalforregulating Tgif1 signaling,although, or to the acidTGFß retinoic pathways,do notbut linkreveal any strong to HPE. has that Tgif1 effectsinmultipledevelopment, mouse someofwhich may attributablebe skeleton etal., (Bartholin 2006;Melhuish 2016).al., Overall,et thesestudiessuggest provideevidence for exencephaliclesswithphenotype, much defects. However,frequent HPE-like did this thebackground, notdefects, primarilyalthough 2006).etHPEal.,(Kuang only Again, theinC57BL6 exencephalyexposedin acid-inducedteratogenecity washigher resultingobserved,alsoain frequency of additional whichinmouse mutant, singleaexon of togethergeneshumanin HPEpatients(El-Jaick 2007; etal.,Shen & 2005). An Walsh, ofto mutationconsistent these genes, with thescarcity ofevidencein mutations both for didnot mice revealanycooperative evidence onfor development effects the ofHPEdue

Author Manuscriptvitroin, werethat partly ondependent altered signaling TGFß (Mar Hoodless,&

Tgif1 Tgif1 Tgif1 nullhaveembryos expressiongene changes and proliferation This article isprotected by copyright. All rights reserved. null anhad incompletelyembryos hypoplasticpenetrant head or Tgif1 as a as HPEcausal gene mouseinmodels. nullembryos and severe defectsmore in theaxial John Wiley&Sons Wiley-Phase 1 Tgif1 Nodal Tgif1 expressionwas shownalter to retinoic gene (Kerr,Cuykendall,gene Luettjohann, null mice (Bartholin et2008; al., Tgif1 Tgif1 was developeddeleted anterior mutantembryos toretinoic Tgif1 and Shh inmutations Tgif1 null

As theAs structuresforebrain begintoat around form ShhE7.75, issignaling responsible (PrCP) underlying (PrCP) ventral the precursorforebrain starting atE7.75.tissue Shh initiatingfordorsoventral patterning.expressionShh inisseen first theprechordal plate Pevny, & McMahon,Pevny,& Ina2002). loxP deletionof sequencesflanked all cells infrom theepiblastby E6.5(Hayashi, Lewis, combininga defectsThe gastrulation embryosinseen lacking canbypassed both Tgifs be by stagewherea even early of precursor forms the HPEphenotypewould apparent. be earlyessential functions embryogenesis,in butdouble mutant embryos do notsurvive to EARLYMOUSEPATTERNING FOREBRAIN beingTgif function requirednormal development.forebrainfor anddefects HPE-like with defects essentiallypenetrance, withconsistent100% overall al., (Powerset 2010; etal.,2012).Taniguchi embryosleft-rightThese had asymmetry (Krzeszinskietal.,2014). combinationThe of both in mutations this model, deletion this model, of a neuroepitheliala expression similar pattern of to that broadlyexpressedthroughout the mouseembryo embryonic and from day 6-8.5, has (E) The structuraland The functionalsimilaritiesbetweenand Tgif1 clearlyTgif2 raise the possibility redundantphenotypes. of During early embryogenesis, ;Tgif2 et al.,2010;Shen et 2005).& Similarly, Walsh, majority ofmajorityembryos withat mutations ofthethree allelesfour ( gastrulationin embryosinfailure werethatnull homozygousboth whereas genes, for the homozygous 2010), although2010), developmentaldefects,and themice were and largely viablenormal etal., (Powers patterns of patterns expressionhightheinis branchialtelencephalon, and arches, somites. expressionThus such asAs HPE.such with oftheirloss function mightbe expectedcontribute anteriorto developmentalto defects the neural platethe head-fold andstages (E7-7.5)(JinByet al.,2006). E9.5, embryopre-streak (E6.0),and thenathigher levelstheinanterior inand at thebudtail +/- Author Manuscriptwere normal) al.,2010). (Powerset and Thus performTgif1 Tgif2 redundant, Tgif1 Tgif2 Tgif1

and Tgif2

nullallele withconditional ina mutation deletion, doublenull embryos the approximatelysurvived to E10.5-11 null were latermice shown tohavebonein defects resorption Tgif2 This article isprotected by copyright. All rights reserved. Tgif1 Tgif1 arelargely overlappingtheinearly embryo whenatstages , of, mutation is drivenis bySox2-Cretransgene a that conditional inresults Tgif2 John Wiley&Sons nullbackground with Sox2-Cremediated Wiley-Phase 1 Tgif2 Tgif1 indidnot cause mice severe expressionseen is throughout the Tgif1 later developmentin (Powers Tgif1 Tgif1 (Powersetal.,2010). In Tgif1 Tgif2 +/- and ;Tgif2 appearsbe to Tgif1 Tgif2 -/- or or

resulted Tgif1 -/- Page 12of31 Page 13of31

Although there isthereAlthoughevidence no HPE-associated for humaninvariants dorsoventralwith gradient higher expressionGli3 dorsally. Joyner,(Fuccillo,Fishell, & In2006).developing neuralthe tissue, isGli3 expressedain has signaling, been shown playrole crucialdorsoventral to ain forebrain patterning The lack ofventrallackThe identity inseen Eto, Motoyama,Nishimura, & 2002;Rallu etal.,2002; Ragsdale,Tole, 2000).& Grove, requirement theproperfor balancebetween anddorsalizingGli3 ventralizing (Aoto,Shh consistentisidentity. withThis expansion an oftheventralsignal,Shh and suggestsa haveembryos a withforebrain dorsally tissue,ventralexpanded lacks that dorsal dose ofdose 1 Shimamura & Rubenstein,&Shimamura 1997). Homozygous diencephalonby E9.0,tissue where specifiesShh identityventral(Geng & 2009;Oliver, PrCPexpression the inis essential activating expressionShhfor theinoverlying ventral FOREBRAINDEFECTSMOUSE IN EMBRYOSBOTH LACKING other patterning, signaling pathways clearly contribute. etal.,2006).(Gutin Shhalthough Thus the pathway majordriverofa is forebrain ventraltelencephalondevelopment, withoutdisruption ofthe signalingShh pathway theinsignalinganterior Fgfr1by deletionandof the Fgfr2 in resultsgenes defective signaling FGF toHPEin humans (Dubourgetal., disruptingInmice,2016). FGF Foxg1ondepend and FGFsignaling, and evidenceisthere linkingaffect that mutations development. additionalThese pathwayscan developmentspecify that forebrain likely andShh Gli3, mustadditionalthere be pathways specify telencephalonthat However,2002).since thedevelops relativelyforebrain absencenormally ofboththe in is criticalfactors dorsoventralforebrainfor patterning et(Aotoal.,2009;Rallu al., et ventricle that lacks ventralventriclelacks that divideidentityand to fails into twohemispheres

of a conditionalof notsurvivedo and aroundbeyondE8.0 (Powers 2010).al., Epiblast-specificet deletion both lacking Tgif genes inhavegastrulation,fail defects the Nodal signaling pathway, and Tgif1share overlappingTgif2 functionsduring development. Mouse embryos E10.5-11. While overallE10.5-11. size largelyandWhile forebrain morphologyare E8.25-9.25, normal at gastrulationdefects,and theseconditional double nullembryos survive to approximately 996). Gli3, a zinc-fingerGli3,996).a transcription factor that aprimarily as repressoractsof Shh

Author ManuscriptreducedisGli3 genetically,suggesting that the antagonismmutualof thesetwo

Tgif1 allele ain This article isprotected by copyright. All rights reserved. Tgif2 nullShhembryos be can partially when rescued the John Wiley&Sons nullbackground allows bypass of for the Wiley-Phase 1 Shhnullembryos have a forebrain homozygousGli3 null TGIF1 TGIF2 AND

(Chiang (Chiang etal., , in , mice TGIF2

E8.25, seen as E8.25, in ventralspecification,including separatefailure ventrallipsto of the thecephalic at folds lackingbothembryos show Tgifs precursorHPE consistent forms of with impaired , which Gli3, normallyis Shh restricting important for signaling (Taniguchietal.,2017). regulate outputTgifs the Shh signalingof through direct transcriptional repressionof pathwayHPE associated (Taniguchietal.,2012). haveMolecular studies shown that expressionin ( etal.,2012).Taniguchi Furthermore, markeranalysis that demonstrates thenasal Nodal-dependentdecrease proliferationinin the al.,neuroepithelium (Taniguchiet 2017; atmesenchyme themidline(Fig. 4A). reductionThis in sizeforebrain toapartlydueis abnormalventral as morphologyforebrain tobisectwiththe ventralfailure a head embryos E10.0 lacking haveTgif a function significantly smaller vesicle,forebrain well as neuralmidbrain tube embryosintoclosefails both lacking Tgifs,evenBy by E9.25. associated with associated andHPE, older rarein embryos lacking both (witha Tgifs 2012). isal., withconsistentThis oftheduplicationlack of midline features facial 2017). Expression2017). of below)mutation, see canonicalmoreHPE phenotypes wereobserved (Taniguchiet al., neuroepithelium.Reducing and modest, aredefects,thereother as such reducedproliferationin the ventral However, it is likelyisHowever, it the reduced both both Tgifs,while dorsal combination ofcombination mutationsbothin todisruptionpart ofthe balanceShh-Gli3al., (TaniguchiHowever,et 2012). the separation better ofthe consistent facialHPE-likewith phenotypefields, the inbeingdue nullinresultsembryos some restorationofnormal development, includingforebrain that also havealsoheterozygousthat a (Taniguchietal.,2017). surprisingly, Perhaps aof embryos fraction lacking both Tgifs suggestingphenotype, independent effects oftwothesepathways regulatedby Tgifs expression in this context.expression this in patterning ofpatterningthe since forebrain, a neuroepithelialproliferationpossiblyand but defects, other notaffect does dorsoventral theexcessreducingNodal responsetheseinembryos partially restores theimpaired classicHPEphenotype (Taniguchi etal.,2017). possible One explanationis this thatfor Author ManuscriptPax7eye)and ( Tgif1;Tgif2

Pax2 Shh Shhreducedisinventral the ofembryos forebrain mouse lacking This article isprotected by copyright. All rights reserved. ) ) fieldstoundergo complete fail separation4B) (Taniguchi (Fig. et null (Fig.3)(Taniguchiembryos etal., addition,2012). theIn expressionGli3increased, suggestingis disruption this ofmajor nullembryos, as theincrease in levels,Gli3 byallele, deletionofonein conditionaldouble Nodal expressionShh is notentirelyhigher due dorsalto Gli3 Nodal Nodal John Wiley&Sons survivemutationlate butto gestation have a Wiley-Phase 1 and mutationnotdoes reducetheexcess appearsGli3 improveto further the expressionGli3relatively is Nodal Gli3

Page 14of31 Page 15of31

and independentrole forallowing in Tgifs normal activationbyGli3 Nodal, expressionrequiredisrestrictto maintainingproliferation of rostral neuroepithelialforebrain afterSooncells.initial the al.,2008;(Silvestri et etal.,2012).Taniguchi Telencephalicsignaling Fgf8 criticalis for Nodaland,signalingembryosin lacking Tgifs,thisexpression increases by E9.0 that removingonethat copy of Tgifs. In addition, Tgifs. In conditionaldoublenull embryoswere that heterozygous bothfor Tgifs appearto Tgifs exert complexon effects telencephalic and regulate dorsoventral regulate patterning ofdeveloping the directlyforebrain, repress Tgifs neuroepithelialproliferation(Fig.5, left).As Shh-Gli3the balanceis established to Nodal response to signaling,which telencephalicinitiatesexpression, Fgf8 limits and pathwayssignaling It observed.is appears arethat required Tgifs dampen to the amelioratedby reducing either orandNodal,Gli3 ofboth Shh disruption Fgfthe and 5). Phenotypes(Fig. in theofembryos lackingforebrain allTgif functioncan be together, Taken molecularand embryological analyses suggest that function Tgif onimpinges signalingmultiple pathways associatedwith early development forebrain argue maythatis this thepathwaycauses HPE that when by of lossdisrupted Tgifs. conditionaldoublenull embryos, and persistentthe moreeffects Shhon the pathway if knownthese effectsofFgf on Tgifs contributesignaling tothephenotypes in induction,Nodaldependent then reducing Gli3-dependent However,repression. is it not telencephalonprogenitors,where Fgf8activates subsequentlycontribute tothereduced (Taniguchipartialetal.,2017). This rescueof proliferation, suggestingproliferation, indirectan bymechanismwhich arepermissive Tgifs normal for patterningdorsoventral togetherwith Nodal adependent improvement in forebrain doublemutantembryos, ventral the forebrain,otherindirectly than PrCP vialargelythe is Gli3. in TgifWhile intact right). One remaining whetheris question regulateTgifs expression,preventingexcess dorsalization, and limiting Fgf8also expression (Fig.5, expression(TaniguchiForebrainetal.,2012). Storm etal.,2006). Storm AtE9.5, embryos lackinghaveboth Tgifs reduced telencephalic Nodal expression,Foxg1 due totheectopic expression Gli3 (Taniguchi etal.,2012). Thus

In addition In toeffectson thepathway,Shh affectsof both loss Tgifs forebrain

Authorpartiallyhad restored Manuscript This article isprotected by copyright. All rights reserved. expressionShhreduced is theinPrCP, and this may does notdoes Gli3 restore expressionShh in ventraltelencephalon E9.5 at John Wiley&Sons Wiley-Phase 1 expressionShh theinventral A forebrain. Gli3- Shh Shhexpression bemay due tobetter Fgf8 expressionShh embryosin lacking expressionFoxg1 (Aotoal.,2002; et expressionsuggestedis by the fact expressioninitially is activatedby Fgf8 expressionShh in theandPrCP Fgf8 expression, limiting first expression to Gli3 Gli3 Fgf8 Fgf8

Nodal andmutations mousein lackingembryos Tgifs.Heterozygous inmutations boththe forebrain difficult difficult to withreconcile theHPEobserved bothin patientswith heterozygous locilikely at these leadtoreducedof output theNodalpathway,signaling whichseems HPEinpatients found (DeCruzLa etal.,2002; etal.,2008).Roessler variantsGenetic FOXH1,whichand part ofthemediates Nodal response,transcriptional have been VariantsHPE.in encodingthe genesNODAL the the ligand, TDGF1co-receptor(Cripto) Evidencehumanstudies from suggests reducedthatNodal signaling can tocontribute SIGNALING,NODAL FUNCTION ANDHPE TGIF excessexpression,Gli3 excess Nodalrather than to assignaling previously suggested. that theHPEthatphenotypes patientsinseen with suggest that independently Tgifs Nodal regulate signalingthe Shh and pathways, and diencephalonand more directlydetermined.Nonetheless,remainstobe thesestudies absent, ratherabsent,than exhibiting a clear HPEphenotype in seen as 1998), is Li, the tissue clearly withofpresumptive distinct most the being tissue forebrain canembryos appearsimilarproboscis-like to the withphenotypeseen &HPE (Nomura et al.,2001;Shawlot et etal.,1999). truncationsinWhile M. Anderson, (R. Lawrence,Stottmann, Bachiller, Klingensmith,& 2002;Mukhopadhyay visceralandendoderm thePrCP,both ofwhich criticalearly are for inductionforebrain indicating lowerthat Nodalassociatedissignaling with impairedanterior formation of whereasphenotypesHPE-like less are common.is withconsistentThis studies micein However,2012).the defects in theseembryos areprimarily truncations,anterior NodalimportantissignalingHPEin pathogenesis & (Nomura Li, 1998; etal.,Taniguchi signaling has beenhassignaling implicated HPEinand Nodalactivatesthat ais relatively this bymechanismrare which HPEdevelops. Givenloss that of FGF8 associated oftenare more with congenitalabnormalities,other perhapssuggesting that Human variantsHuman in embryos lackingbothembryos byheterozygous aTgifs etSilvestrial.,2008). onBased the rescueofproliferationdefects and survival mouse of pathwayto do rise give that HPEso might byFGF8 affecting et (Dubourg 2016; al., ittelencephalon,is speculatetempting to lossthat of infunction mutations theNODAL and expression.Shh However, whether regulate Tgifs

Author ManuscriptSmad2

miceingenescan resultHPE,in againsuggesting that reductionina NODAL , This article isprotected by copyright. All rights reserved. FOXH1 and John Wiley&Sons Wiley-Phase 1 TDGF1 Nodal TGIF1 inare relatively HPErare patients, and mutation, it ismutation, it bealso possiblethat variants bemay primarily to due Nodal;Smad2 expressionShh theinPrCP Fgf8 Shh expression thein mutant mouse null embryos. TGIF1

Page 16of31 Page 17of31

ACKNOWLEDGEMENTS Anderson, 2010). AlthoughAnderson,2010). ciliahave defects not demonstratedbeen theindeveloping

Work in theWork labis by Wotton R01,funded authorsThe thankA.Carlton,Melhuish T. commentsA. Shahand on for themanuscript. pathogenesis. losssuggeststhat of Tgif function alsomaypathway into this feed HPEcontribute to to that clear thesignalingShh pathway disruption,majortarget a is and for work recent potential and genetic functionalinteractionsbetween the isHPE-causalknowngenes, it Mizugishi,Nakata, Mikoshiba,Aruga,& Despite2001). theobvious complexities of interactionswith and Gli SMAD transcription (Houtmeyersfactors etal.,2016;Koyabu, notedshouldbe ZIC2alsothat has linked been toandbothShh Nodalviasignaling TGIF1 al.,2008). Recentet evidence suggestsa similar transcriptionaldirect activation of humans, E.Anderson etal.,(A. 2017). Interestingly, other frequentlyHPE mutated in genes on ciliafunction couldreducetheShh theresponseinas in seenforebrain, culturedcells offorebrain mutantembryos,Tgif mouse itremainspossible that effectsofloss of Tgif SHH SIX3 wasSIX3 showndirectly to activate required required themajorityShhfor signalingof Anderson et al.,E. (A. 2017;Goetz & evidencesuggeststhatalso regulate Tgifs ofprimary the formation cilia, whichare topromoterrepress expression (Taniguchietal., etal.,2012).2017; Taniguchi Recent Gli3 balance is disturbedbalanceisGli3 embryosin lackingandbinds Tgifs, directlyTgif1 to the the Despitemultiple causesof HPEhumansin andit interestingmice,is tonote apparent the convergence Shhon the pathway.signaling above,discussedShh- As the primarilytoaltereddueShh signaling. isTGIF at function least partly independentofpathway, this with defectspatterning signalingNODAL itresultHPE,inis likely HPE most thatseen the inabsence ofthe environmentalinsults (Taniguchietal.,2017). However, variantssince inof activators uncoveringthereby patterning defectsHPE anddue additionalto orgenetic Nodalsignaling reduced allow might severelymore a embryo toaffected survive, and expression by ZIC2 (Ishiguro,Hatayama, Aruga,2018).&Otsuka, However, it SIX3 Author ManuscriptSIX3 and each positively expression regulate oftheother etal., (Geng 2008;Jeong

ZIC2 This article isprotected by copyright. All rights reserved. , may also link,alsomay eitherfunctionallyShh to signaling or to SHH John Wiley&Sons Wiley-Phase 1 expression theinmouseventral and forebrain,

NS077958D.W.).(to TGIF1 Gli3 . .

Anderson,A.E., Hao,Taniguchi,K.,Melhuish,Y., Shah,A., A., D.,T. Turner, S. . . . Anderson, R. M., Anderson,R. Lawrence, A.R., Stottmann, R. Bachiller, W., Klingensmith,&D., J. Aoto, K., Shikata,Y.,Aoto, Imai,Matsumaru, H., D., Tokunaga, Shioda,T., Motoyama,S., . . . Aoto, K., Nishimura,Aoto, K., T.,Motoyama, & Eto, (2002).J. Mouse regulatesFgf8GLI3 REFERENCES Bartholin, L., Bartholin, Melhuish, A., Powers,T. S.E., Goddard-Leon,S., Sutherland, Treilleux,I., Burglin, T. R.Burglin,(1997). AnalysisT. TALEsuperclasshomeobox of (MEIS,PBC,genes Evans, R. M.,&Evans, R. Mangelsdorf,D. J. Nuclear (2014). Receptors, RXR, BigandBang. the Bertolino, E., Bertolino, Reimund, &B., D., Clerc,R. (1995). novelWildt-Perinic, A homeobox L., Bartholin, Powers,S.Melhuish, E., Lasse,S.,A., M.,T. & D. Weinstein, Wotton, El-Jaick, K.B., El-Jaick, Powers,Bartholin,E., S. Myers,K.R.,L., Hahn, J., M.,...Orioli, I. Chiang, C., Litingtung,Chiang,C.,Y., Lee,E., Young, K.E., Corden, L.,J. & Beachy,H., Westphal, &R.,Burglin, Affolter,T. HomeodomainM. (2016). anproteins: update. Bertolino, E., Bertolino, Richards,S., Clerc,&G.,R. Wildt, (1996). Expression G. of novela murine Chinnadurai, G. (2002). an unconventionalCtBP, transcriptional corepressorin Dubourg, C., Dubourg, Carre, Hamdi-Roze,Mouden,H., W., Roume,C., J., Abdelmajid,B., . .. De La Cruz,La De M., Bamford,J.N., R. Burdine, R.Roessler,D., E., Barkovich,A.J., Chinnadurai, G. (2007). Transcriptional regulationbybindingC-terminal proteins. A. E., &E., D. A. (2008).MaternalWotton, is Tgif required vascularization for of the Celldoi:10.1128/MCB.00527-16 Biol,37(5). D. Wotton,(2017). Repress Expression and Tgif1 Tgif2 oftheEvi5l.RabGAP (2), 320-332.Biol, 251(2), expressionapoptosisand theindeveloping neural limb tube, and face, bud. in the the inmouse. Chordin (2002). nogginpromoteand organizing centersof developmentforebrain and craniofacialand morphogenesis. Mouse (2009). J. isShh required prechordalforplate maintenance during brain embryonicplacenta. animals. animals. KNOX, Iroquois, TGIF) revealsnovela conservedbetween domain plants and (1), 255-266.Cell, 157 (1), doi:10.1016/j.cell.2014.03.012 responsivemotif. which protein recognizescore a TGT and withfunctionally interferes a retinoid- (2006). inhibitsretinoidTGIF signaling. holoprosencephaly. M.Muenke, (2007).Functional analysis in of mutations associated TGIF with doi:10.1002/humu.23038 hedgehog function.gene A.(1996). CyclopiaP. defectiveand axialmiceinlacking Sonic patterning 497-521. 125(3), doi:10.1007/s00412-015-0543-8 proliferation. homeoboxindevelopinggene cerebellarthe external layer duringgranular its developmentoncogenesis.and a NewMajorSignalinga Pathway. David,(2016).Mutational V. in Spectrum Holoprosencephaly is ShowsThat FGF 422-428. 110(5), Authorofdomainassociated is TDGF1 with human defects.forebrain .Muenke,Donnai,D., . . loss-of-functionM.A (2002). mutation theinCFC CellBiochem Biol,39 Manuscript

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database. Asterisksdatabase. indicatevariants havethat altered in function Boxednonsense). variantsaffect codons show that variation no the ingnomAD shown,using theindicated color coding(Blue:missense, Red: frameshift, Black: shownB)aboveSequencebelow and each. variantsin HPETGIF1patients are from recruitmentCtBPacid motif isin that found TGIF1butnotamino-acid An TGIF2. isscale areinpresentRD) both. red boxThe HD amino-terminal to the represents amino the five carboxyl-terminalregion itand (+20), to thecarboxyl-terminal repressiondomain (C-ter shownbetween.Major are shown:homeodomain features 20(HD),The acid amino the shownschematicallyidentitythe percentwith similarityconservedand the for domains 1.Variants Figure human TGIF1.in LEGENDS Figure 2. Figure ofModels transcriptionalregulation byTGIFs. shown.also Fordatabase.missense percentagevariants, the arewithinthat homeodomain the is those identified those in Tgif2. 3.Defective Figure development neural tube inmouse embryos andTgif1lacking conclusivelybeen ruled out. a to consensusTGIF sitethisin acase, requirement DNAforbinding byhasTGIFs not limitingactivationinteraction, (minussign). Although no nois therebinding evidence for expressionactivate gene (plus sign).beindirectly can recruited TGIFs via SMAD At C) TGFß-responsiveSMAD genes, bind proteins toSMAD elements(SBE)binding to which (RXR), activates expressiongenewhen (plusligand sign) ain bound NRcomplex. site, consensus orby indirect via recruitment interactionwith the retinoid receptorX byreceptors. nuclear The recruitment of canbyTGIF1 be binding tothe TGIF can TGIF1 be todirectrepeatrecruited (DR)hormone responseelements(HRE) bound important).more inresultsThis repression(negativelinked ofa sign) B) target gene. DNAconsensusvia sitewell (cTGTCAa,a defined with thecentral bases beingfive Sequencevariation in althoughassays, notall variantsshown have been here tested functionally. C) each typeofvariantaffectingeach thecoding sequenceof

Scanningelectron microscopy (SEM) images oforcontrol mutant mouseembryos Author Manuscript

TGIF1 This article isprotected by copyright. All rights reserved. TGIF1 from HPEpatients, from and both for fromthegenes gnomAD and TGIF2 John Wiley&Sons A) andhumanThe TGIF1 proteinsareTGIF2 Wiley-Phase 1 is shownis summary inform. ofnumbers The TGIF1 A) can TGIFs bind directlyto and TGIF2 orvitroincellbased areshown for

Whole mount Whole the the vesicle.forebrain Scale 500 bars: at10.0dpc.embryos whiteThe linesindicatethe planeofthecoronal sectionsthrough andimages hematoxylin eosin(H&E)and stained sections ofcontrol coronal cdKOand bars: 100µ bars: indicated the dayspost coitum (dpc).Views anterior are from the of embryo.Scale the epiblast(cdKO, for conditionalspecific double of knock-out Figure 4. separationFigure Midline defectsabsence in the ofTgifs. primarilysimplicityis schematic.for of the longerNodalno isexpressed. The separationoftemporal effectsonTgif NodalGli3and shownearlier schematic.the inReduced proliferation later,continuemay butby ~E9.0, effects additionto on theindicated genes/proteins,Nodalinhibition ofproliferationis indicatesShh afrom Tgif possibleindirect permissive ofShh on effect Tgifs signaling. In positivelines indicatewithregulation, red bars inhibitory effects. dashed The arrow to panellefthandThe represents ~E8.0and thehandright ~E9.0. Arrows(green) indicate interactionssignalingdiscussed here both (Tgif plusrepresents Tgif1 Tgif2 function). headearly shownismouse schematicallyleft), (anterior the to togetherwith the major view).250µ Scalebars: embryos,showingexpression ofPax7 (above, view) frontal Pax2and (below, ventral Figure 5.Tgif regulationFigure ofsignaling inthedeveloping forebrain. PLoSGenet.(2012), 8(2):e1002524.

Author ManuscriptImagesm. etal,from Taniguchi PLoS 8(2):e1002524. Genet. (2012),

inhybridization situ ofstage-matchedimages dpc (~10.0 for control) This article isprotected by copyright. All rights reserved. m m for andPax7 200µ John Wiley&Sons µ Wiley-Phase 1 m whole-mountandm for 200 m m for Pax2 . . Images etal,from Taniguchi Tgif1 A) Whole-mount and µ m m for B)sections. A side viewthe of Tgif2 ) areshown ) at Page 26of31 Page 27of31

sequence of TGIF1 sequenceTGIF1 of amino acid CtBP recruitment motif that is found inis recruitment found CtBP acid TGIF1motifamino that An TGIF2. amino- not but repression domain (C-ter RD) are presentrepressionare The in both. (C-ter RD) domain redamino- box from the gnomAD database. the gnomAD the from missense For variants, withinare the percentagethat is homeodomain also based assays, although although hereassays, not based shown allhavevariants variation Sequence been in testedC) functionally. The homeodomain (HD), the (HD), homeodomain regionacidThe 20amino carboxyl-terminal carboxyl-terminal the it to (+20), and Figure 1. Variants in humanhuman The FigureA) TGIF1. TGIF1Variants TGIF21. proteins with schematically and shown are variation in the gnomAD database. in gnomAD variation the altered indicate Asterisks that have function variants in in vitro or cell the percent identity and similarity for the conserved domains shown between.percent the Major shown identity shown:similarityare conservedfor the features and domains TGIF1 and TGIF2 is shown in summary form. form. numbers inTGIF2 summary isThe shown TGIF1 and variant type of affectingeach of coding the and below each. B) B) below and Sequenceineach. TGIF1 from variants HPEshown, are patients usingindicated the color

Author Manuscriptnonsense). codingBlack: (Blue: frameshift, missense, Red: affect showcodons variants Boxed no that

and TGIF2 are shown for those identified forthose TGIF2shown are from and patients,genes forboth HPE and in TGIF1 This article isprotected by copyright. All rights reserved. 170x104mm(300 300DPI) x John Wiley&Sons Wiley-Phase 1 shown. shown.

terminalrepresents HD the to five the acid above isscale shown

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