cilia and ciliopathies in congenital heart disease

Cilia and Ciliopathies in Congenital HeartDisease

Nikolai T. Klena, Brian C. Gibbs, and Cecilia W. Lo

Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh,Pennsylvania 15201

Correspondence: [email protected]

A central role for cilia in congenital heart disease (CHD) was recently identified in a large-scale mouse mutagenesis screen. Although the screen was phenotype-driven, the majority ofgenes recovered were cilia-related, suggesting that cilia play a central role in CHD patho-genesis. This partly reflects the role of cilia as a hub for cell signaling pathways regulatingcardiovascular development. Consistentwith this, manycilia-transducedcell signalinggeneswere also recovered, and genes regulating vesicular trafficking, a pathway essential for cilio-genesis and cell signaling. Interestingly, among CHD-cilia genes recovered, some regulateleft–right patterning, indicating cardiac left–right asymmetry disturbance may play signifi-cant roles in CHD pathogenesis. Clinically, CHD patients show a high prevalence of ciliarydysfunction and showenrichment for de novo mutations in cilia-related pathways. Combinedwith the mouse findings, this would suggest CHD may be a new class of ciliopathy.

Congenital heart disease (CHD) is one of themost common birth defects, found in an

estimated 1% of live births (Hoffman andKaplan 2002). With advances in surgical pallia-tion, most patients with CHD now survive theircritical heart disease such that currently thereare more adults with CHD than infants bornwith CHD each year (van der Bom et al.2012). However, CHD patient prognosis is var-iable, with long-term outcome shown to be de-pendent on patient intrinsic factors rather thansurgical parameters (Newburger et al. 2012;Marelli et al. 2016). This is likely driven by ge-netic factors, given CHD is highly associatedwith chromosomal anomalies (Fahed et al.2013), and with copy number variants (Gless-ner et al. 2014). In addition, CHD has been

shown to have a high recurrence risk, with fa-milial clustering indicating a genetic contribu-tion (Gill et al. 2003; Oyen et al. 2009). Theidentification of the genetic causes of CHDmay provide mechanistic insights that canhelp stratify patients for guiding the therapeuticmanagement of their clinical care.

Investigations into the genetic causes ofCHD in human clinical studies have been chal-lenging given the high degree of genetic diver-sity in the human population. This has made acompelling case for pursuing the use of a sys-tems genetic approach with large-scale forwardgenetic screens in animal models to investigatethe genetic etiology of CHD. Although manyanimal models have provided invaluable in-sights into the developmental regulation of car-

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diovascular development, investigations intothe genetic etiology of CHD must be conductedin a model system with the same four-chambercardiac anatomy that is the substrate of humanCHD. The mouse is one such model system,advantageous not only given its similar four-chamber cardiac anatomy, but also inbredmouse strains are readily available with ge-nomes that are fully sequenced and annotatedthat would facilitate mutation recovery. More-over, cardiovascular development in the mouseembryo is well studied, providing a strong foun-dation to interrogate the developmental and ge-netic etiology of CHD.

DEVELOPMENT OF THE CARDIOVASCULARSYSTEM

Congenital heart defect is a structural birth de-fect arising from disruption of cardiovasculardevelopment. Formation of the four-chamberheart in mammals is orchestrated by the highlycoordinated specification and migration of dif-ferent cell populations in the embryo that to-gether form the complex left–right asymmetricanatomy of the cardiovascular system. In themouse embryo, ingression of cells through theprimitive streak at E7.5 generates the anteriormesoderm forming the cardiac crescent–con-taining cells of the first heart field (FHF) andadjacent to it, the second heart field (SHF) (Fig.1) (Buckingham 2016). Cells of the FHF mi-grate toward the midline, fusing to form thelinear heart tube at E8.0 (Fig. 1). Pharyngealmesoderm located anterior and medially con-tinues to be added to the expanding heart tube,as the heart tube undergoes rightward loopingat E8.5, delineating the primitive anlage of theleft ventricle (LV) (Fig. 1). This is followed byaddition of SHF cells to the anterior and poste-rior poles of the heart tube, giving rise to theoutflow tract (OFT), right ventricle (RV), andmost of the left and right atria (LA, RA) (Fig. 1).

Normal development of the heart also re-quires the contribution and activity of severalother extracardiac cell lineages, including thecardiac neural crest cells derived from the dorsalhindbrain neural fold. The cardiac neural crestcells migrate into the cardiac OFT in two spiral

streams, helping to remodel the pharyngeal archarteries and orchestrating OFT septation toform the two great arteries—the aorta and pul-monary artery (Kirby and Waldo 1990). Thepharyngeal endoderm and ectoderm also playan important regulatory function in develop-mental patterning of the aortic arch arteriesand the OFT. Dynamic processes mediating en-docardial epithelial–mesenchyme transforma-tion (EMT) lead to formation of the cushionmesenchyme that provides early valve functionin the embryonic heart. These endocardial cush-ion tissues later remodel to form the matureleaflets of the outflow semilunar and atrioven-tricular valves (Fig. 1). Another extracardiac cellpopulation required for heart development arethe pro-epicardial cells that originate near theseptum transversum. These cells migrate to theheart via the sinus venosus, delaminating ontothe surface of the heart, and forming the epicar-dium that plays an essential role in developmentof the coronary arteries. Together, these diversecell populations are recruited to orchestrate for-mation of the mammalian heart, an organ thatis an unexpected mosaic of distinct cell lineages.

FOUR-CHAMBER HEART—THEANATOMICAL SUBSTRATE FORCONGENITAL HEART DISEASE

The cardiovascular system in mouse and humanis adapted for breathing air, being comprised offour chambers organized into functionally dis-tinct left versus right sides. This allows the for-mation of a separate pulmonary circuit thatpumps deoxygenated blood from the body tothe lungs via the RV and a systemic circuitpumping oxygenated blood from the lung tothe body via the LV. This left–right asymmetricorganization is critically dependent on appro-priate patterning of the left–right body axis andentails formation of an atrial and ventricularseptum separating the right versus left sides ofthe heart. This allows for compartmentalizationof the heart into four chambers, LA versus RAand LV versus RV. This is coupled with septationof the OFT into two great arteries, the aorta,which is inserted into the LV and pulmonaryartery into the RV, and formation of the atrio-

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Cilia and Ciliopathies in Congenital Heart Disease

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ventricular and outflow valves that allow unidi-rectional blood flow. It is this complex left–right asymmetric developmental patterning ofthe cardiovascular anatomy that ensures effi-cient oxygenation of blood with air exchangevia the lungs. The perturbation of this distinctfour-chamber cardiac anatomy in CHD invari-ably results in neonatal mortality unless surgicalintervention is provided to palliate the struc-tural heart defects. Identifying the genetic caus-es of CHD may help elucidate the developmen-tal processes contributing to CHD and suggestsnew avenues for prevention or intervention.

CENTRAL ROLE OF CILIA INCARDIOVASCULAR DEVELOPMENT ANDCONGENITAL HEART DISEASE

To elucidate the genetic etiology of CHD, alarge-scale, near-saturation level forward genet-ic screen with ethylnitrosourea (ENU) chemicalmutagenesis was conducted (Li et al. 2015b).This phenotype-driven cardiovascular screenused fetal echocardiography, a noninvasive im-aging modality routinely used clinically forCHD diagnosis (Fig. 2). This allowed high de-tection sensitivity and specificity for CHD di-agnosis and allowed the recovery of a wide spec-trum of CHD in the mouse screen similar tothose observed clinically (Figs. 2 and 3) (Liuet al. 2014). From ultrasound screening of�100,000 mouse fetuses, we recovered .200mutant mouse lines with a wide variety of CHD.

Using exome-sequencing analysis, �100CHD-causing mutations were recovered in 61genes, with more than half being cilia-related(Fig. 2) (Li et al. 2015). The cilia genes recoveredincluded proteins localized in the cilia transitionzone, basal body/centrosome, ciliary axoneme,and also multiprotein complexes in the cyto-plasm required for cilia assembly (Fig. 2). Mostof the proteins recovered are expressed in bothmotile (9þ 2) and primary cilia (9þ 0), such ascomponents of the cilia transition zone. How-ever, some genes encode proteins unique to mo-tile cilia, such as the motor dyneins Dnah5 andDnah11 localized in the outer dynein arm re-quired for motile cilia function (Fig. 2). Manyof these cilia protein components are known to

cause various human ciliopathies, mostly in-volving nonmotile, primary cilia defects, suchas in Joubert syndrome (JBTS), Jeune syndrome,nephronophthisis, Meckel–Gruber syndrome,and others. The motile cilia mutations recoveredin the screen are linked to the sinopulmonarydisease primary ciliary dyskinesia (PCD). Al-though CHD is not an essential feature of cilio-pathies, it is notable that the mutants we recov-ered were all based on having CHD phenotypes.

Further indicating the important role of cil-ia in CHD pathogenesis, we also recovered mu-tations in 12 CHD genes that are in cilia-trans-duced cell signaling pathways, including genesmediating sonic hedgehog (Shh), transforminggrowth factor b and bone morphogenetic pro-teins (TGF-b/BMPs), and Wnt signaling (Fig.2). This enrichment of genes mediating cell sig-naling reflects the central role of cilia as a hubfor signal transduction pathways essential to theregulation of key cardiovascular developmentalprocesses. Also unexpected was the recovery of10 CHD genes involved in vesicular trafficking.This included Dynamin 2 and Ap2b1 requiredfor clathrin-mediated endocytosis, adaptin pro-teins Ap1b1 and Ap2b1, and Lrp1, Lrp2, andSnx17 mediating endocytic receptor recycling(Li et al. 2015b). Significantly, vesicular traffick-ing plays an essential role in cilia biology, withciliogenesis initiated with capture of a ciliaryvesicle by the mother centriole followed bydocking of the basal body to the cell membraneand fusion of additional secondary vesicles thatallow lengthening of the ciliary axoneme (So-rokin 1962; Kobayashi and Dynlacht 2011; Reiteret al. 2012). Vesicular trafficking and receptorrecycling also play important roles in the regu-lation of cell signaling. Although the endocyticpathway was not previously known to play a rolein CHD, its importance can be easily appreciat-ed in the context of its role in regulating cilio-genesis and cilia-transduced cell signaling.

CILIA AND CILIA-TRANSDUCED CELLSIGNALING IN HEART DEVELOPMENT

The overall finding that the large majority of theCHD genes recovered were cilia or cilia-relatedwas unexpected, given the screen was entirely

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phenotype-driven. Hence, they point to a cen-tral role for cilia biology in regulating cardio-vascular development and the pathogenesisof CHD. Primary cilia in the developing heartwere first identified via electron microscopy inthe chick, rabbit, mouse, and lizard embryos(Rash et al. 1969). These were observed onlyin nonmitotic cardiomyocytes or myoblasts,

whereas in the adult heart tissue, cilia wereonly observed in fibroblasts. A more recentstudy of the mouse embryo showed that ciliacan be found throughout the early E9.5 hearttube (Slough et al. 2008). As developmentprogresses to E12.5, cilia continue to be ex-pressed in the atria and in the trabeculated myo-cardium (Fig. 3J). Cilia are also found in the

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Figure 2. Congenital heart disease (CHD) mutants recovered from mouse mutagenesis screen by fetal echocar-diography show preponderance of cilia-related mutations. Vevo 2100 color flow Doppler imaging showed criss-cross pattern of blood flow indicating normal aorta (Ao) and pulmonary artery (PA) alignment (A) confirmedby histopathology (B). E16.5 mutant (line b2b327) showed blood flow pattern indicating single great artery (PA)and ventricular septal defect (VSD) (C), suggesting aortic atresia with VSD, confirmed by histopathology (D).Color flow imaging of E15.5 mutant (line b2b2025) with heterotaxy (stomach on right) showed Ao/PA side-by-side with Ao emerging from right ventricle (RV) (E), indicating double outlet right ventricle (DORV)/VSD (F)and presence of atrioventricular septal defect (AVSD) (G,H ). Histopathology also showed bicuspid aortic valve(BAV) (I), interrupted aortic arch (IAA) (J ), and common atrioventricular (AV) valve (K). (Bottom) Diagramssummarize genes recovered causing CHD that are related to cilia or cell signaling, providing biological context ofCHD gene function. Color highlighting indicates CHD genes recovered; asterisks denote CHD genes recoveredfrom previous screen (Shen et al. 2005). R, Receptor; TGN, trans-Golgi network (adapted from data in Li et al.2015b).




atrial endocardial layer and more prominentlyin the endocardial cushion mesenchyme (Fig.3K,L) and in the epicardium (Slough et al. 2008;Willaredt et al. 2012; Li et al. 2015b). A numberof cilia-transduced cell signaling pathways havebeen shown to play essential roles in regulatingcardiovascular development and may contrib-ute to the pathogenesis of CHD. These include

Shh, TGF-b, BMP, and Wnt signaling. Fourgenes involved in Shh signaling were recoveredfrom the mouse CHD screen, including Sufu,Fuz, Tbc1d32, and Kif7 (Fig. 2). Also recoveredwere six genes involved in TGF-b /BMP signal-ing, including Cfc1, Megf8, Tab1, Ltbp1, Smad6,and Pcsk5 and three mediating Wnt signaling—Ptk7, Prickle1, and Fuz (Fig. 2).

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Figure 3. Congenital heart defects in a Wdpcp mutant and cilia localization. (A–H) Episcopic confocal histo-pathology showed a WdpcpCys40 mutant with an incomplete septum unevenly dividing the outflow tract (OFT)into one large and one small chamber, indicating pulmonary atresia (PAtr; black arrow in B). Also observed wasan atrioventricular septal defect (AVSD; asterisk in D). Shown in A and C are comparable views of a control heart.In wild-type hearts (E), cardiomyocytes were observed in the OFT cushion (arrow), but inWdpcpCys40mutants,cardiomyocytes were mostly absent in the cushion tissue (asterisk in F). Cardiomyocytes in outflow cushion ofwild-type embryos (G) visualized with MF20 immunostaining showed polarized cell morphology with distinctelongated finger-like projections (asterisks) aligned with direction of cell migration and projecting into formingoutflow septum (arrow in G). In contrast, in WdpcpCys40mutant embryos (H ), the cardiomyocytes showedrounded morphology without obvious cell polarity, nor the elongated cell projections seen in wild-type em-bryos. (From Cui et al. 2013; reprinted under the Creative Commons CCO public domain dedication.) (I–M)Immunofluorescence staining of cilia with acetylated tubulin (green) and g-tubulin (red) antibodies (from datain Cui et al. 2013). Shown are the detection of cilia in the mouse embryonic node (I), and in the myocardium(J ), outflow (OFT) cushions (K), and atrioventricular cushions (L) of wild-type E12.5 embryonic mouse heart.In contrast, cilia are missing in the atrioventricular cushion tissue of Cc2d2a mutant known to develop atrio-ventricular septal defect (adapted from data in Li et al. 2015b). Scale bars, 100 mm (E, G).

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Role of Shh Signaling in Cardiac Developmentand CHD

Shh signaling is the best-described cilia-trans-duced cell signaling pathway. Numerous studieshave shown that ablation of cilia can result in adrastic reduction of Shh signaling (Huangfuet al. 2003; Han et al. 2008; Goetz and Anderson2010). During heart development, Shh is ex-pressed in the pharyngeal endoderm and inthe foregut endoderm adjacent to incomingSHF derivatives in the dorsal mesenchyme pro-trusion (Dyer and Kirby 2009). Shh knockoutmice show atrial and atrioventricular septationdefects, defects in OFTseptation, and abnormalpharyngeal arch artery patterning (WashingtonSmoak et al. 2005). The outflow septation de-fects are characterized by the aorta shifted right-ward overriding the septum, and with eitherpulmonary atresia or a hypoplastic pulmonaryartery observed in conjunction with a variabledegree of ventricular hypertrophy. This constel-lation of defects is reminiscent of tetralogy ofFallot (TOF), one of the most common complexCHD observed clinically (Washington Smoaket al. 2005). Using Cre targeted deletion analy-sis, it was shown that these outflow defects re-flect a dual requirement for pharyngeal endo-dermal-derived Shh in the cardiac neural crestcells and the SHF derivatives (Goddeeris et al.2007). These studies showed Shh signaling tothe SHF and cardiac neural crest cells are re-quired for OFT septation, but not for eitherOFT lengthening, or cushion formation, respec-tively. As the Shh knockout embryos showed areduction in the number of SHF derivatives, thissuggested a requirement for Shh in the specifi-cation of the SHF (Hildreth et al. 2009).

Role of Wnt Signaling in CardiacDevelopment and CHD

Primary cilia also play a role in the transductionof canonical and noncanonical Wnt signaling(Clevers 2006; MacDonald et al. 2009; Walling-ford and Mitchell 2011; May-Simera and Kelley2012) pathways that are also essential for nor-mal heart development. One early evidencelinking cilia with b-catenin-dependent canoni-

cal Wnt signaling was the observation thatknockdown of basal body components bbs1,bbs4, and bss6 resulted in several-fold increasein Wnt activity in zebrafish (Gerdes et al. 2007).The functional link between Wnt signaling andcilia was also shown by the observed localiza-tion of noncanonical Wnt/planar cell polarity(PCP) components, such as Inversin, Dishev-elled, Vangl2, and Wdpcp in the basal bodyand/or ciliary axoneme (Fig. 4A,B) (Montcou-quiol et al. 2003; May-Simera and Kelley 2012;Cui et al. 2013). Other studies also showed a rolefor cilia as a switch that can constrain canonicalversus noncanonical Wnt signaling (Ross et al.2005; Simons et al. 2005; Barrow et al. 2007;Gerdes et al. 2007; Corbit et al. 2008; Huangand Schier 2009; Stottmann et al. 2009; Lien-kamp et al. 2012; Oh and Katsanis 2013). How-ever, the precise mechanism by which ciliaregulate Wnt signaling is not well understood.

In mice, the noncanonical Wnt/PCP genessuch as Celsr, Frizzled3 (Fzd3), Fzd6, Vangl1-2,and Dvl1-3 are highly expressed in the OFT(Etheridge et al. 2008; Paudyal et al. 2010).Mice with mutations in the PCP genes Vangl2,Scrib (Phillips et al. 2007), Dvl 1, 2, and 3(Hamblet et al. 2002; Etheridge et al. 2008;Sinha et al. 2012), Wdpcp (Cui et al. 2013),and Pk1 (Gibbs et al. 2016) show a spectrumof CHD phenotypes involving OFT malalign-ment and septation defects, such as double out-let RV (Fig. 2E,F), overriding aorta, pulmonaryatresia (Fig. 3B), and persistent truncus arteri-osus (Henderson et al. 2006; Cui et al. 2013;Boczonadi et al. 2014; Gibbs et al. 2016). Thesecardiac defects likely reflect a role for nonca-nonical Wnt/PCP pathway in regulating thepolarized migration of cardiac neural crest andSHF derivatives (Tada and Smith 2000; Mont-couquiol et al. 2003; Simons et al. 2005; Verziet al. 2005; Cohen et al. 2007; Simons and Mlod-zik 2008; Schlessinger et al. 2009; Gibbs et al.2016). Consistent with this, examination ofmouse embryonic fibroblasts derived from theWdpcp or Pk1 mutant embryos showed inabilityof the cells to polarize and engage in directionalcell migration (Figs. 4C–G, 5K–N). In contrastto Shh deficiency, Wnt/PCP disruption causedfailure of the OFT to appropriately lengthen





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Figure 4. Wdpcp is a cilia protein regulating cell polarity, directional cell migration, and the actin cytoskeleton.(A,B) IMCD3 cells immunostained with Wdpcp (green) and acetylateda-tubulin (red) antibodies show Wdpcplocalization in the axoneme and ring-like structure (arrowhead) at the ciliary base. Localization of Wdpcp (red)in this ring-like structure is better seen with a 3D isosurface reconstruction, which also shows some colocaliza-tion of Septin-2 (green) with the Wdpcp ring. (C–G) In a wound-healing assay, control mouse embryonicfibroblasts (MEFs) (A) show good alignment with the direction of wound closure (indicated by white arrow). Incontrast, WdpcpCys40 mutant MEFs (B) showed a disorganized distribution. These differences in cell polaritywere also reflected in the Golgi orientation (white line drawn through the center of the Golgi stained green)(E,F). In wild-type MEFs, the Golgi (green) was mostly situated at the cell’s leading edge (E,G), aligned with thedirection of wound closure (white arrow), whereas the WdpcpCys40 mutant MEFs show randomized Golgiorientation (F,G). Scale bars, 20 mm (A, B, C, D, F). (H–N) Confocal imaging of Sept2 (red) and Wdpcp(green) showed they are colocalized in actin stress fiber (phalloidin stained, blue) in wild-type MEFs (H–J),but in the WdpcpCys40 mutant MEFs, Wdpcp expression was lost (blue, L), whereas Sept2 immunostaining (red,K,M) showed the loss of colocalization with actin (blue) (K,L,M). (L) Wdpcp (green) is enriched at the cellcortex where actin filaments (phalloidin) insert into vinculin (red)-containing focal adhesions (N) in wild-typeMEFs. (Adapted from Cui et al. 2013 under the Creative Commons CC0 public domain dedication.)

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A B C +/–

D

I

J

FE

HG

K L

m/m

TZ

+/– m/m

+/– m/m

+/–

+/+

M N+/+

m/m

m/m

V+/+ = 1.367±V–/– = 1.657±

p = 0.0017

D+/+ = 0.513±0

0 20 40 60 80 0 20

Migration orientation (degrees)

40 60 80

Per

cent

age

1

2

3

4

5 +/+p = 0.0261

m/m

D–/– = 0.523±p = 0.7864

m/m

m/m

Apical

+/+

Apical

TZ

Figure 5. Shortened outflow tract (OFT) and defects in cell polarity and directional cell migration in the Pk1Bj

mutant. (A–D) E10.5 Pk1Bj mutant embryo. B and D show shortened OFT as compared with that of hetero-zygous embryo (C,D). (E–H) Islet1 immunostaining show distribution of SHF cells in the dorsal pericardialwall of the OFTof control (E,G) and Pk1Bj mutant embryos (F,H). Magnified views of region denoted by arrow-heads in E and F revealed a cuboidal (H ) rather than flat squamous (G) epithelial morphology in the homo-zygous mutant versus heterozygous embryo. (I,J ) b-Catenin (green) and laminin (red) antibody staining ofwild-type (I) and Bj mutant embryos (J ) shown in the E10.5 Bj mutant embryo, marked disorganization of theepithelium in the transition zone (TZ) of the pericardial wall where SHF derivatives are found. Confocal imagingshowed laminin (red) is localized basally (arrowhead I) in the TZ of the control embryo, but in the mutantembryo, it is localized apically (arrow) and basally (arrowhead J ), indicating a loss of normal epithelial polarity.The distribution of b-catenin (green) remains at the cell surface in both the control and Bj mutant embryos.(K,L) Myocardiolization defect in the OFTof Pk1Bj mutants. Examination of the striated banding pattern fromMF20 immunostain showed the developing myofilaments in the heart are closely aligned and oriented towardthe direction of myocardialization in the wild-type E14.5 embryo (K), but in the Bj mutant, the myofilamentsare sparse and are largely oriented perpendicular to the direction of myocardialization and septum formation(L). (M,N) Wound closure assay shows a defect in directional cell migration in Pk1Bj mutant mouse embryonicfibroblasts (MEFs). The migration path of MEFs 8 h after wound scratch were well aligned with the direction ofwound closure, but tortuous paths were observed with increased velocity for the Pk1Bj mutant MEFs (M,N)(adapted from data in Gibbs et al. 2016). Scale bars, 0.5 mm (A, B); 50 mm (E); 20 mm (K).




(Fig. 5A–D). In the Pk1 mutant, the epithelialorganization and apical-basal polarity of theSHF derivatives in the OFT are disrupted. Thiswould suggest a defect in convergent-extensioncell movement required for delamination of acohesive epithelial sheet mediating OFT length-ening (Fig. 5E–J). This is followed later by amyocardialization defect of the OFT (Figs.3E–H, 5K–L), that together with the shortenedOFT likely account for the great artery mala-lignment defect in the Pk1 mutant.

Role of TGF-b Signaling in CardiacDevelopment and CHD

A role for cilia in mediating TGF-b signalingwas recently shown with the finding that ligandbinding causes accumulation of TGF-b recep-tors at the base of the cilium, in a region knownas the ciliary pocket (Clement et al. 2013). Thistriggers receptor-mediated endocytosis involv-ing clathrin-coated vesicles, leading to down-stream activation of SMAD phosphorylation(Clement et al. 2013). The essential role ofTGF-b/BMP signaling in CHD is well de-scribed via in vitro and in vivo analyses of chickand mouse embryos, and also with the exami-nation of knockout mouse models (Combs andYutzey 2009; de Vlaming et al. 2012; Kruithofet al. 2012; von Gise and Pu 2012). These studiesshow TGF-b/BMP signaling has multiple rolesin cardiovascular development that include theregulation of both endocardial EMT and endo-cardial cushion development (Potts and Run-yan 1989; Camenisch et al. 2002). For example,early endocardial cushion development to ac-quire critical valve-like function requires BMPsignaling in cardiac neural crest cells via theBMPRIA receptors (Nomura-Kitabayashi et al.2009). A role for Tgfb2 in OFT and aortic archremodeling is indicated by the finding thatTgfb2 knockout mice die perinatally with dou-ble outlet RV and interrupted aortic arch (San-ford et al. 1997).

The disturbance of TGF-b/BMP signalingis likely to play a major role in the valvular de-fects seen in mice harboring mutations disrupt-ing clathrin-mediated endocytosis and endo-cytic receptor recycling (Ap2b1, Dnm2, Ap1b1,

Snx17, LRP1, LRP2). These endocytic mutantsall show OFT malalignment and endocardialcushion defects, phenotypes reminiscent ofthose observed in mutants with disruption ofTGF-b/BMP signaling (Li et al. 2015b). Simi-larly, mutations affecting cilia integrity in theendocardial cushions may cause disruption ofcilia-transduced TGF-b/BMP signaling re-quired for normal valve development. Thus,mutation in Cc2d2a, a cilia transition zonecomponent, causes selective loss of cilia in theatrioventricular (AV) but not outflow cushions,and as might be expected, such mutants showedAV valve defects, while the outflow valves werespared (Fig. 3K–M).

ROLE OF CILIA IN SPECIFICATIONOF CELL POLARITY AND POLARIZEDCELL MIGRATION

Some cilia proteins may help regulate cardio-vascular development through cross talk, di-rectly or indirectly, with the cytoskeleton tospecify cell polarity and directional cell migra-tion, morphogenetic cell movements, and epi-thelial–mesenchyme cell transformation. Giv-en the basal body is a microtubule organizingcenter that can regulate nucleation and organi-zation of microtubule outgrowth, one conceptthat has emerged is that cilia may regulate thecytoskeleton through dynamic interactionswith PCP components and, in this manner,specify cell polarity and polarized cell migration(Figs. 4 and 5) (Wallingford and Mitchell 2011;May-Simera and Kelley 2012). These dynamiccell processes may help to direct the long-dis-tance migration of multiple extracardiac cellpopulations to the embryonic heart that are re-quired for normal heart development. This in-cludes cells from the SHF, neural crest cells, andthe pro-epicardial cells. In addition, cilia direct-ed reorganization of the actin cytoskeleton mayalso contribute to the regulation of EMT, suchas required for the emergence of cardiac neuralcrest cells from the dorsal neural fold, endocar-dial EMT mediating formation of the cardiaccushions and valves, or epicardial EMT thatgenerate the epicardially derived cells formingthe coronary vessels. These developmental pro-

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cesses involving dynamic reorganization of thecytoskeleton is impacted by cilia and, in con-junction with cilia-transduced cell signaling,may help orchestrate development of the car-diovascular system.

Although the role of cilia in the regulation ofcell polarity and directional cell migration in thecardiovascular development is well described inthe context of OFT morphogenesis (see above),the precise mechanism and role of the cilia inmodulating cell polarity is less understood. Inthis regard, it is worth pointing out that Wdpcp,a PCP component also known as Fritz, is local-ized not only in the cilia, but it is also colocal-ized with septins in the cilia (Kim et al. 2010;Cui et al. 2013) and in the actin cytoskeleton(Kim et al. 2010; Cui et al. 2013). In mouseembryonic fibroblast (MEF) cells deficient inWdpcp, a marked reorganization of the actincytoskeleton is observed (Fig. 4H–L), and thisis associated with altered focal contacts (Fig.4N) inability to establish cell polarity and en-gage in directional cell migration (Fig. 4C–G).Similar studies of MEFs harboring a mutationin the PCP component Pk1 also showed a sim-ilar loss of cell polarity and defect in directionalcell migration (Fig. 5M,N) (Gibbs et al. 2016).Together, these findings suggest that cilia muta-tions may cause CHD not only via the disrup-tion of cilia-transduced cell signaling, but ciliamutations also may disrupt the cytoarchitectureand perturb the establishment of cell polarity,polarized cell migration, and/or EMT.

CILIA IN LEFT–RIGHT PATTERNINGAND CONGENITAL HEART DISEASE

The enrichment of cilia genes was also notablein that it included a subset of genes that causedCHD in conjunction with left–right patterningdefects. This likely reflects the known require-ment for cilia in left–right patterning, with pre-vious studies indicating that motile cilia at theembryonic node is required to break symmetry(Fig. 3I) (Hirokawa et al. 2009; Nakamura andHamada 2012). Analysis of motile cilia mutantmice revealed CHD is typically observed in con-junction with heterotaxy, the randomization ofleft–right patterning (Tan et al. 2007). This is

consistent with the well-described clinical asso-ciation of complex CHD with heterotaxy (Linet al. 2014). As the heart is the most left–rightasymmetric organ, and this asymmetry is essen-tial for efficient oxygenation of blood, it is per-haps not surprising that left–right patterningdefects may play a major role in CHD patho-genesis.

Among 34 cilia mutations recovered causinglaterality defects, 22 genes perturbed the prima-ry cilia (Cc2d2a, Anks6, Nek8, Mks1, Cep290,Bicc1) versus 12 genes that disrupted motile cil-ia (Dnah5, Dnah11, Dnai1, Daw1, Armc4,Ccdc151, Drc1, Ccdc39, Dyxc1x1, Dnaaf3) (Liet al. 2015b). The latter genes are known tocause PCD, a ciliopathy that is autosomal reces-sive (Collins et al. 2014; Horani et al. 2014; Loboet al. 2015). In PCD, immotile/dyskinetic ciliain the airway cause mucociliary clearance de-fects that can lead to severe sinopulmonary dis-ease. Approximately half of PCD patients showsitus solitus, half situs inversus totalis, and vary-ing numbers up to 8% may show CHD withheterotaxy (Kennedy et al. 2007; Shapiro et al.2014). The disturbance of laterality with PCDreflects the essential role of motile cilia in left–right patterning. Studies in the PCD mutantmouse models showed each PCD mutationcan give rise to three phenotypes—approxi-mately half with situs solitus or situs inversusand half with heterotaxy, with complex CHDobserved only with heterotaxy (Tan et al.2007). Although the heterotaxy mutants mostlydie prenatally or neonatally from the CHD, mu-tants with situs solitus or inversus are largelyviable postnatally without CHD. Videomicros-copy showed most of these PCD mutants haveimmotile cilia in the embryonic node, even ashalf of the mutants show normal or invertedconcordant situs that indicate the breaking ofsymmetry.

These striking observations suggest thatmotile cilia are not absolutely required forbreaking symmetry, nor for left–right axis spec-ification, although motile cilia are clearly re-quired for high-fidelity situs solitus specifica-tion. As CHD is only seen with heterotaxy,this provides a clue that patterning of the car-diovascular system may occur very early in de-





velopment, at the time the left–right body axisis specified. Even as these findings show thatmotile cilia play an important role in left–rightpatterning, the recovery of 24 mutations affect-ing primary cilia suggests nonmotile cilia alsoplay an essential role in laterality specification(Li et al. 2015b). Previous studies suggested atwo-cilia hypothesis in which motile cilia at thenode generated right to left flow (for additionalinformation, see Shinohara and Hamada 2016).This is proposed to trigger mechanosensorytransduction of primary cilia in the perinodalcrown cells, causing left-sided calcium releasethat is propagated into the surrounding lateralplate mesoderm, causing the breaking of sym-metry (Nonaka et al. 2002; McGrath et al. 2003;Bruekner 2007; Yoshiba et al. 2012). However,this model has been called into question recent-ly given the failure to detect cilia-mediated me-chanosensation and calcium release (Dellinget al. 2016).

A role for primary cilia in left–right pattern-ing could be easily understood neverthelesswithout invoking mechanosensation, becauseShh and TGF-b signaling, both cilia-transducedpathways, play important roles in left–right pat-terning. Although Shh knockout mice do notshow overt laterality defects, they show LAisomerism (Hildreth et al. 2009). Furthermore,the single outflow vessel seen in the Shh knock-out mouse is said to represent pulmonary atre-sia, as the single great artery shows Pitx2c, indi-cating a left-sided identity (Washington Smoaket al. 2005). It is interesting to note in chickembryos where Shh plays a much more primaryrole in left–right patterning, the experimentalmanipulation of left–right expression of Shhcan cause CHD, confirming its importance ofleft–right patterning in the pathogenesis ofCHD (Levin et al. 1995). Signaling mediatedby the TGF-b family of growth factors, includ-ing nodal, lefty1, and lefty2, are well describedto specify the left–right axis. This nodal signal-ing cascade is believed to propagate left–rightspecification initiated at the node. How muta-tions affecting primary cilia may contribute tothe disruption of left–right patterning is notknown, but it is thought to cause disturbance inthe propagation of this nodal signaling cascade.

CILIARY DYSFUNCTION AND CILIOMEMUTATIONS IN CHD PATIENTS

The unexpected enrichment for mutations incilia-related (ciliome) genes and genes involvedin endocytic trafficking and in cilia-transducedcell signaling (Shh, WNT/Pcp, TGF-b) in themouse mutagenesis screen point to a centralrole for cilia in CHD pathogenesis. To assessthe relevance of these findings to human CHD,we investigated the findings from exome-se-quencing analysis of CHD patients by the Pedi-atric Cardiac Genomics Consortium (PCGC)(Zaidi et al. 2013). In this analysis, the focuswas on examining de novo predicted pathogeniccoding variants. Although the PCGC publica-tion focused on the recovery of de novo variantsin a number of chromatin-modifying genes,interestingly, we noted among the 28 de novodamaging mutations identified in the PCGCCHD patient cohort, 13 or nearly half were ingenes associated with pathways identified in themouse forward genetic screen—that is, ciliogen-esis, endocytic trafficking, and cilia-transducedcell signaling (SHH, WNT, TGF-b) (Table 1),with LRP2 being a gene recovered in both thePCGC CHD patients and the mouse CHD mu-tants recovered in our screen. We also noted therecovery in the PCGC cohort of a de novo vari-ant in Pitx2, a gene known to play an essentialrole in left–right patterning, supporting an im-portant role for left–right patterning distur-bance in CHD pathogenesis.

Further supporting a central role for cilia inthe pathogenesis of CHD are clinical studiesshowing a high prevalence of ciliary dysfunctionin CHD patients (Nakhleh et al. 2012; Garrodet al. 2014). Given that respiratory compli-cations are among the biggest postsurgicalcomplications for CHD patients, we previouslyhypothesized that some CHD patients with res-piratory complications may have undiagnosedPCD. These studies were initiated with an exam-ination of CHD patients with heterotaxy. Nasalscrapes were conducted and video microscopywas used to examine cilia motility in the nasalepithelium. This analysis showed a high preva-lence of ciliary dysfunction in CHD patientswith heterotaxy. The ciliary motion defects

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observed span a spectrum that included someshowing dyskinetic ciliary motion to slow oreven immotile cilia. Overall, .40% of the pa-tients showed ciliary dysfunction (Nakhleh et al.2012). Although this was associated with an en-richment for coding variants in PCD genes, nopatient was either homozygous or compoundheterozygous for any PCD gene mutations.Thus, although CHD patients with heterotaxyare at high risk for ciliary dysfunction, these pa-tients largely do not have PCD. Since this initialstudy, a large study has been conducted compris-ing .200 patients with CHD of a broad spec-trum, mostly without heterotaxy. This analysisshowed a similar high prevalence of ciliary dys-function and this was correlated with increasedrisk of having PCD-related respiratory symp-toms (Garrod et al. 2014). Together, these find-ings suggest ciliary dysfunction is commonly as-sociated with CHD in the human population.

Although these studies focused on assessingmotile cilia function, we note many cilia genesare expressed in both motile and primary cilia.Hence, the high prevalence of ciliary dysfunc-tion in CHD patients may reflect not only theperturbation of motile cilia genes, but also genesrequired for primary cilia function. Indeed, werecently showed a patient harboring compoundheterozygous mutations in WDR35 causing

Sensenbrenner syndrome, a ciliopathy thoughtto affect only the primary cilia, showed motilecilia dysfunction. Pulmonary function assess-ments indicated obstructive airway disease thatsuggested possible mucociliary clearance de-fects in the airway (Li et al. 2015a). Indeed, sev-eral clinical studies have shown an increase inrespiratory symptoms and disease in patientswith other ciliopathies thought to affect onlythe primary cilia, indicating the distinction be-tween ciliopathies involving motile versus pri-mary cilia may not be so clear cut (Tobin andBeales 2009). These findings suggest furtherstudies are warranted to assess ciliopathy pa-tients of a wide spectrum for potential pulmo-nary complications, especially for those whowill undergo high-risk surgeries, such as thoseinvolving cardiopulmonary bypass.

CONGENITAL HEART DISEASE ANDCILIOPATHIES

It is notable that many cilia genes recovered inthe mouse forward genetic screen for CHD-causing mutations are genes clinically knownto cause various human ciliopathies. This in-cludes not only motile cilia genes associatedwith PCD, but also cilia genes linked to variousciliopathies thought to affect the primary cilia,

Table 1. Functional annotation for 13 PCGC patients with de novo mutations

Patient ID CHD typea Gene Mutation Gene function annotation

1-00638 CTD FBN2 p.D2191N TGF-b signaling1-02020 HTX SMAD2 p.IVS12 þ 1G . A TGF-b signaling1-02621 HTX SMAD2 p.W244C TGF-b signaling1-00197 LVO BCL9 p.M1395K Wnt signaling1-01828 CTD DAPK3 p.P193L Wnt signaling1-01138 LVO USP34 p.L432P Wnt signaling1-00802 LVO PTCH1 p.R831Q Shh signaling/ciliome1-02598 HTX LRP2b p.E4372K Shh signaling/endocytic trafficking1-01913 Other RAB10 p.N112S Endocytic trafficking1-00750 LVO HUWE1 p.R3219C Ciliome1-01151 CTD SUV420H1 p.R143C Ciliome1-00853 CTD WDR5 p.K7Q Ciliome1-02952 LVO PITX2 p.A47V Laterality-related

Based on exome-sequencing analysis of congenital heart disease (CHD) patients by Pediatric Cardiac Genomics

Consortium (Data from Zaidi et al. 2013).aCTD, Conotruncal defect; HTX, heterotaxy; LVO, left ventricular obstruction.bLRP2 is an endocytic gene also recovered from our mouse screen.





such as in JBTS, polycystic kidney disease, acro-callosal syndrome, hydroelethalus, Leber con-gential amaurosis, Meckel–Gruber syndrome,Bardet–Biedl syndrome, etc. (Li et al. 2015b).While in our mouse screen, ciliopathy geneswere recovered based on mutations causingCHD phenotypes, clinically these ciliopathiesare not commonly associated with CHD. Thismay reflect ascertainment bias given that thepatient population represent only human fetus-es that can survive to term and, hence, are lesslikely to have severe cardiac anomalies. Indeed,clinical studies of aborted or stillborn fetuseshave shown that the human fetal populationhas more than ten times higher incidence ofCHD as compared with those in the clinical pa-tient population (Hoffman and Kaplan 2002).Consistent with this, most of the CHD ciliop-athy mutants recovered from our screen wereinviable to term and were harvested pretermafter in utero phenotyping by fetal echocardiog-raphy. On the flip side, there is undoubtedlyascertainment bias in our screen in the recoveryof mutations in ciliopathy genes that specificallycan cause CHD. That different ciliopathy mu-tations may have varying levels of penetrancefor CHD phenotypes is suggested by observa-tions of our mutant Hug (Damerla et al. 2015).This mutant has a mutation in Jbts17, a geneencoding a cilia transition zone protein knownto cause JBTS (Srour et al. 2012). Hug mutantsshow cerebellar defects expected for JBTS andthey also can show CHD comprising of pulmo-nary atresia. However, the CHD phenotype isincomplete in penetrance, as some Hug mutantsshow no heart defects (Damerla et al. 2015).These observations suggest that different muta-tions in the same ciliopathy gene may generatedifferent phenotypic outcome and this perhapscan be further modified by the genetic back-ground of the individual.

In light of these observations, we suggestthat, clinically, CHD may be considered a struc-tural birth defect related to ciliopathies. Howev-er, unlike other ciliopathies, which are relativelyrare (,1 in 10,000) and with a Mendelian re-cessive inheritance, the much higher prevalenceof CHD (up to 1%) and its sporadic occurrencewould suggest the contribution of cilia-related

or ciliome genes in CHD will be multigenic andhighly genetically heterogeneous. Such complexgenetics is expected to reflect the complexity ofcilia biology in which sequence variants foundamong different “ciliome” genes may affect thefunction of large multiprotein complexes thatregulate ciliogenesis and cilia structure andfunction. Given that there are hundreds of cil-iome genes that contribute to cilia assembly andcilia structure and function, it is perhaps notsurprising that CHD patients are observed tohave a high prevalence of ciliary dysfunction.While the CHD genes recovered from the mousescreen were by design recessive mutations, weexpect mutations in these same genes can con-tribute to more complex genetic models ofdisease. Such complex genetics may also con-tribute to classic ciliopathies, as there are clinicalreports of PCD patients and patients with otherciliopathies that have no homozygous or com-pound heterozygous ciliopathy mutations, butinstead show multiple heterozygous mutationsin known PCD or other ciliopathy genes (dePontual et al. 2009; Li et al. 2016). A future chal-lenge is to develop an effective bioinformaticspipeline for modeling and interrogating suchcomplex genetics and assess the contributionof ciliome mutations in the pathogenesis ofCHD and other structural birth defects.

CONCLUSIONS

CHDs are the most common structural birthdefects, and despite its prevalence, the geneticetiology of CHD remains poorly understood.Interrogations into the genetic landscape forCHD using a large-scale forward genetic screenin mice unveiled a central role for ciliome genesin the pathogenesis of CHD. These studies sug-gest the perturbation of cilia and cilia-trans-duced cell signaling pathways may play a centralrole in the pathogenesis of CHD. The futurechallenge is to clinically translate these findingsin mice to patients with CHD. The finding ofa high prevalence of ciliary dysfunction in CHDpatients and the enrichment of de novo patho-genic variants in cilia and cilia-related pathwaysin CHD patients would suggests such studieswill be fruitful and may provide the basis for

N.T. Klena et al.




stratifying patients to optimize the clinicalmanagement of patient care. The recent findingof primary cilia in the endothelial cells of theaorta regulating anti-atherosclerotic responsesalso point to a potential role for cilia in adultcardiac disease (Dinsmore and Reiter 2016).Further work in the future will be needed toclarify the role of cilia biology in human CHDand perhaps other cardiovascular diseases, andwith such insights may come new avenues oftherapeutic intervention to improve the out-come for patients with critical heart disease.

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