miRNA-processing enzyme Dicer is necessary forcardiac outflow tract alignment and chamber septationAnkur Saxenaa,b and Clifford J. Tabina,1

aDepartment of Genetics, Harvard Medical School, Boston, MA 02115; and bDivision of Biology, California Institute of Technology, Pasadena, CA 91125

Contributed by Clifford J. Tabin, November 9, 2009 (sent for review October 4, 2009)

MicroRNAs (miRNAs) have previously been implicated in a numberof developmental processes, including development of the ven-tricular myocardium of the heart. To determine what, if any,additional roles miRNAs play in cardiogenesis, we deleted themiRNA-processing enzyme Dicer specifically in the developingmurine heart. Embryos lacking cardiac Dicer lived longer thanreported in previous studies using different alleles to removecardiac Dicer activity and displayed a highly penetrant phenotypeof double outlet right ventricle with a concurrent ventricularseptal defect. Before the defect’s onset, Pitx2c and Sema3c, bothrequired for outflow tract morphogenesis, were up-regulated inDicer-deficient hearts. Interestingly, mesenchymal apoptosis in theoutflow tract normally required for outflow tract alignment wasgreatly decreased in the mutants, likely contributing directly to theobserved phenotype. In sum, we demonstrate here a specific de-velopmental process, that of outflow tract morphogenesis, beinghindered by the deletion of miRNAs during cardiogenesis.

Congenital heart malformations, resulting frommistakes in thecomplex process of cardiac development, represent the most

common types of defects in newborns with at least 1% of allabnormalities at birth classified as congenital heart defects(CHDs) (1, 2). Over the last few years much progress has beenmade in elucidating genetic pathways underlying cardiogenesis,including the identification of a large number of transcriptionfactors necessary for orchestrating the process. At the same time,there has been an increasing awareness of the importance ofposttranscriptional mechanisms regulating cellular processesduring embryogenesis, including those mediated by the approx-imately 20–25 nt noncoding regulatory RNAs known as micro-RNAs (miRNAs) (3, 4). Although the functions of several specificmiRNAs have been explored in the context of cardiac develop-ment, the full extent to which this class of regulatory moleculeinfluences heart formation remains to be determined.An important strategy for establishing the range of devel-

opmental events regulated by miRNAs is to broadly interferewith all miRNA processing, either throughout the embryo or invarious specific tissues and stages of embryogenesis. This can beaccomplished, for example, through inactivation of the miRNA-processing enzyme Dicer, required for the production of maturemiRNAs from pre-miRNAs (5). Previously reported targeteddeletion of Dicer in mice demonstrated lethality in homozygous-null embryos by approximately embryonic day 7.5 postfertiliza-tion (e7.5) (6), an early time point that precluded investigation ofDicer’s role in cardiogenesis. A more recent removal of Dicerfunction specifically within Nkx2.5-expressing cardiac progenitorcells resulted in developmental anomalies in the heart includingan underdevelopment of the ventricular myocardium and peri-cardial edema, ultimately leading to cardiac failure and embry-onic lethality (7). Cardiac-specific Dicer deficiency was alsoengineered at a later stage under the control of a promoter forthe cardiomyocyte structural protein alpha myosin heavy chain(α-MHC) (8). This later-stage loss of miRNA processing resultedin misexpression of cardiac contractile proteins, disruption of thesarcomeric structure, and a consequent impairment of cardiacfunction. These mice rapidly developed dilated cardiomyopathy,heart failure and postnatal lethality.

These studies highlighted the information to be gained byremoving Dicer function at different stages of development andmaturation of an organ. Accordingly, we decided to furtherexplore miRNA function during heart development by removingDicer activity at a stage in between those reported in the prior twostudies. To accomplish this, we also made use of a Cre transgenedriven by Nkx2.5 regulatory sequences; however, wemade use of adifferent allele (9) than the one previously used by Zhao et al. (7,10). This transgene is also activated from the cardiac crescent stageonwards but is known to be expressed with slightly different spa-tiotemporal kinetics. Crossing these mice with a floxed Dicer al-lele (11) produced Nkx2.5-CreCre/+;Dicerflox/flox embryos lackingmiRNAs in the developing heart. As hoped, due to differences intiming and breadth of expression in the twoNkx2.5-Cre constructsand perhaps also the particular strains of mice used, the survivalperiod of these cardiac Dicer-deficient mice was longer, althoughthey still died in utero. The longer time window allowed us toobserve the effect of loss of miRNA activity on outflow tract ro-tation and septation, events that occur too late to be visualized inthe earlier study using an alternate Nkx2.5-Cre allele and too earlyto be affected in the study using an α-MHC-Cre.Proper formation of the outflow tract (OFT), which gives rise

to the aorta and pulmonary artery, is critical for the division ofoxygen-rich and -poor blood that optimizes cardiac efficiency.How the OFT septates, rotates, and aligns itself with the heart’sventricles has been extensively studied in chicken and mouseembryos, and a large number of targeted deletion mouse modelsdisplay OFT defects (2, 12–14). Understanding how these arise isparticularly relevant as in humans over 20% of CHDs includeproblems in formation or positioning of the OFT and connectingmajor arteries. These include, among other malformations,double-outlet right ventricle (DORV) wherein both the aortaand pulmonary artery exit the right ventricle and persistenttruncus arteriosus (PTA) in which the OFT fails to septate.Although OFT defects can arise in a variety of ways, one

important mechanism involves failure in the process of pro-grammed cell death. The occurrence of apoptosis during OFTmaturation is thought to be crucial for OFT rotation andshortening to properly occur (15–17). Correlation between de-creased programmed cell death and OFT defects has beendemonstrated in mouse targeted-deletion models such as that ofFoxp1−/− (18). However, potential miRNA-mediated regulationof these processes has not been previously explored.Here, weuse aDicer loss-of-function approach to investigate the

roles of miRNAs in OFT development and demonstrate the dis-ruption of critical programmed cell death during OFT morpho-genesis. Two genes, Pitx2c and Sema3c, known to play crucial rolesin this process are aberrantly expressed in theOFT-forming region,consistent with their contributing to the observed phenotype.

Author contributions: A.S. and C.J.T. designed research; A.S. performed research; A.S. andC.J.T. analyzed data; and A.S. and C.J.T. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence should be addressed. E-mail: tabin@genetics.med.harvard.edu.

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ResultsCardiacDeletionofDicerResults inDORVandConcurrentVSD.Dicerflox/flox

embryos were harvested atmultiple time points to ascertain potentialcardiacdefects andembryonic lethality.Table1 illustrates thenumberof embryos collected and genotyped at each time point. Nkx2.5-CreCre/+;Dicerflox/flox mice proved unable to survive beyond e13.75, alater time point of lethality than had been previously reported (7)using an alternatively constructed Nkx2.5-Cre (10). Thin, improperlycompacted ventricular myocardiumwas observed in mutant embryos(arrow, Fig. 1H, compare withG) similar to that previously reported(7) and may contribute to the embryonic lethality. We observed nosignificant increase in cell death in the incorrectly forming myocar-dium of Dicer-deficient embryos. Meanwhile, previous analysis hasshown that this myocardial phenotype correlates with and may beexplained by changes in expression of several structural proteins (7).Although the cardiac chambers underwent basic morpho-

genesis, by e13.0–e13.5 hearts homozygous for the deletion ofDicer demonstrated 79% penetrance (15 of 19 examined em-bryos) of an apparent cleft-like division where a connection be-tween the left ventricle and aorta would normally have existed(Fig. 1 B and D; asterisks). Histological analysis of serial sectionstaken through the heart revealed that the right ventricle wasconnected to both the pulmonary artery and aorta (Fig. 1 F andH, respectively) in a demonstration of DORV, with a concurrentventricular septal defect (VSD) (Fig. 1H, asterisk). DORVleaves no direct outlet for oxygen-rich blood in the left ventricle,and hence it is forced into the right ventricle through the VSD.Neither Nkx2.5-Cre+/+;Dicerflox/Dicerflox littermates norNkx2.5-CreCre/+;Dicerflox/+ littermates displayed any aberrantphenotypes. Thus, complete removal of Dicer activity in theheart is required for the observed cardiac defects to manifest.

Altered Gene Expression During Outflow Tract Development FollowingDicer Deletion.Recent predictions suggest that as much as 20–30%of vertebrate genomes may be influenced by miRNA regulation(19). To attempt to pinpoint specific genetic causes of the ob-served DORV/VSD defect, we undertook a candidate gene ap-proach using RNA in situ hybridization for several genes known tobe important for heart development. Most of the genes tested (seeMethods for list) showed no change in expression levels in Dicer-deficient hearts as compared to wild-type littermates. The ho-meobox transcription factor Pitx2c, however, demonstrated asignificant, site-specific increase in RNA levels in the absence ofDicer at e10.0–e10.5 in the OFT and adjacent ventricular wall(Fig. 2A and B, arrowheads) that continued as late as e12.5–e13.0(Fig. 2 C–F). Pitx2c is known to play an important role in the es-tablishment of OFT positioning relative to the ventricles, and itsabsence leads to defects in the OFT including a significant per-centage of DORV (20, 21). Intriguingly, ectopic myocardial Pitx2expression in iv/iv mutant mice—demonstrating cardiac loopingrandomization and an assortment of subsequent defects—hasbeen shown to correlate specifically with DORV (22), reminiscentof the Pitx2c up-regulation and DORV phenotype shown here.In trying to understand the molecular consequences of altered

Pitx2c expression, we noted previous work that has demonstratedits regulation of Semaphorin3c (Sema3c) in the OFT myocar-dium. Sema3c is a signaling molecule known to play a variety ofroles including regulating axonal guidance, cell survival, andmigration of neural crest cells (23, 24), and mice lacking Sema3cmanifest a PTA phenotype in the OFT (25).

It has been shown that although the targeted deletion ofmurine Pitx2c does not affect Sema3c expression levels at e10.5or e11.5, by e12.5 Sema3c mRNA is nearly completely absent inPitx2c−/− outflow tracts (20). These data suggest a role for Pitx2cin maintaining appropriate levels of Sema3c in the developingOFT myocardium. Consistent with this possibility, we found thatSema3c RNA expression was markedly up-regulated at e12.5 inthe OFT (Fig. 2 G and H, arrowheads) and adjacent ventricularwall (Fig. 2H, arrows) in Dicer-deficient embryos as compared towild-type littermates. These were locations where we had ob-served the earlier up-regulation of Pitx2c, suggesting that Pitx2c

Table 1. Nkx2.5-CreCre/+;Dicerflox/+ × Nkx2.5-Cre+/+;Dicerflox/flox crosses

Stages e10.25–11.0 e11.5–12.0 e12.5–13.0 e13.5 e13.7–14.0

Nkx2.5-CreCre/+;Dicerflox/flox /total 11/47 (23%) 5/17 (29%) 32/125 (26%) 8/33 (24%) 3/29 (10%)*

*Embryos were observed dying from e13.7 onward; those found alive were close to expiration.

Fig. 1. Cardiac deletion of Dicer results in DORV and concurrent VSD.Hearts at e13.0 and e13.5 days postfertilization are from Nkx2.5-Cre+/+;Di-cerflox/flox (wild-type) (A and C) and Nkx2.5-CreCre/+;Dicerflox/flox (mutant) (Band D) littermates. Note the lack of a LV-OFT connection in the mutanthearts (asterisk). Representative sections are shown at e13.0 of RV-OFTconnections (E and F) and LV-OFT connections (G and H). Arrow in (H) in-dicates abnormally thin ventricular myocardium and asterisk indicates VSD.LV, left ventricle; RV, right ventricle; OFT, outflow tract.

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activation may partially contribute to the observed DORV/VSDvia up-regulation of Sema3c.

Programmed Cell Death During Outflow Tract Alignment Is Disrupted.To determine the mechanistic causes of the DORV/VSD, funda-mental processes that play important roles in OFT maturationwere examined. We failed to find any difference in cell pro-liferation, as assayed by H3B staining, between mutant and wild-type littermate embryos at e12.0, e12.5, or e13.0. Similarly, usingTUNEL staining for apoptosis, we observed no significant differ-ence between wild-type and mutant OFT during early time points.Inmice, apoptosis in theOFThas been previously characterized asbeginning around e12.5 and peaking a day later (17).We observeda similar patternwithno apparent apoptosis before e12.5.At e12.5,small numbers of stained cells were observed with no significantdifference betweenwild-type andmutant embryos (Fig. 3A andB).By e13.0 and e13.5, however, widespread apoptosis was evident inwild-type OFT tissue (Fig. 3 C and E), but the number of stainedcells was greatly decreased in Dicer-deficient OFTs (Fig. 3 D andF). Cellular morphology, as well as costaining for muscle actin,suggested that the cells normally undergoing apoptosis at thesetime points in wild-type embryos were predominantly mesen-

chymal cushion tissue. Staining of serial sections demonstratedincreased Sema3c RNA expression in mutant embryos to be di-rectly adjacent to the area of decreased cell death at e13.0 (Fig. 3GandH), suggesting that increased levels of Sema3cmay help inhibit

Fig. 2. Modified gene expression post-Dicer deletion. Pitx2c mRNA levelsare up-regulated in mutant hearts in comparison with wild-type littermatesfrom as early as e10.25 (A and B) through e12.7 (C and D), and e13.0 (E andF). Arrowheads in (B) and (D) indicate up-regulation in outflow tract andadjacent ventricular myocardium. By comparison, Sema3c expression is firstseen up-regulated at e12.5 in mutant hearts (H) in comparison with wild-type littermates (G). Arrowheads in G and H indicate outflow tract ex-pression; arrows in H indicate ventricular expression. LV, left ventricle; Ao,aorta; PA, pulmonary artery; OT, outflow tract.

Fig. 3. Programmed cell death during outflow tract alignment is disrupted.TUNEL-positive cells (green) andmuscle actin costainmarking cardiomyocytes(red) in outflow tract mesenchyme at e12.5 (A and B), e13.0 (C and D), ande13.5 (E and F). TUNEL-positive cells were few in number in bothwild-type (A)and mutant (B) OFT tissue at e12.5, prevalent in wild-types at e13.0 and e13.5(C and E), andmarkedly reduced in mutants at e13.0 and e13.5 (D and F). Sideby side comparison of Sema3c mRNA and outflow tract apoptosis (G andH) inmutant embryos shows Sema3c expression directly adjacent to the area ofdecreased cell death. (I) Quantitation of mesenchymal apoptosis at e12.75–e13.0 in wild-type and mutant OFT. Bars indicate 95% C.I. *P < 0.001.

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mesenchymal apoptosis. Quantitative analysis at e12.75–e13.0 re-vealed a nearly fivefold decrease in mesenchymal cell death incomparison with wild-type OFT tissue (Fig. 3I).

DiscussionThe relatively recent discovery of miRNAs in the early 1990s andsubsequent studies since then have dramatically altered ourunderstanding of gene regulation. However, the small size ofmiRNAs, unclear binding specificities, and other factors havemade it difficult to pinpoint specific functions for individualmiRNAs. While work in this field is now progressing rapidly,here we chose to directly determine the relevance of miRNAs tocardiogenesis by deleting one of the necessary components ofmiRNA processing, the enzyme Dicer.One of the initial conditional deletions of Dicer, done in the

developing limb mesoderm, demonstrated no aberrations inbasic patterning of the limb, analogous to the intact basic pat-terning observed here in the heart. However, although the limbdeletion exhibited greatly increased cell death (11), the Dicerdeletion detailed herein led to decreased cell death, essentiallythe reverse effect. The difference may, in part, be explained bythe fact that in the limb mesenchyme, the increase in cell deathcan be attributed to a late, nonspecific mitotic defect (11). Incontrast, the decrease in cell death we observe is confined to asmall subset of cardiac tissue and exhibits spatiotemporal spe-cificity, suggesting a specific effect mediated by changes in generegulation during OFT development.Interestingly, the expression of many molecular markers was

not perturbed (at least at the level observable by in situ hybrid-ization) in the absence of Dicer activity and hence miRNAprocessing. This stands in contrast to the extremely large numberof genes predicted to be miRNA targets, up to 1/3 of those in thegenome (26–28), but is consistent with the previous observationthat many miRNAs are expressed in reciprocal cell populationsfrom those expressing their target mRNAs (29, 30). Such a di-chotomy of RNA and miRNA expression may act to providerobustness to developing systems rather than major regulation ofgene activity within domains of expression. The same may largelybe true for the molecular pathways orchestrating cardiogenesisas indicated by the fact that many aspects of the heart formednormally in this and other studies in which Dicer activity wasremoved. Nonetheless, as our analysis affirms, some miRNAtargets are critical for normal cardiac formation.The observed aberrant expression of genes known to be

required for proper OFT development, although other testedgenes remained unaffected, points to a precise effect. Pitx2c andSema3c will certainly not be the only affected genes in a broaddeletion of Dicer, but it is intriguing to speculate on their par-ticular roles in regulating levels of apoptosis during OFT align-ment. The removal of Smad4 signaling in cardiac neural crestcells (CNCCs) has been suggested to non-cell autonomouslydown-regulate Sema3c and other molecules in non-CNCC OFTin concert with defects such as PTA and tract elongation (31),illustrating the crosstalk among multiple cell types necessary forOFT formation. In addition, Sema3c has a known capacity topromote cell survival such as that demonstrated with non-cell

autonomous effects on cerebellar granule neurons and endo-thelial cells in vitro (32, 33). These and other data lend a clue asto how up-regulation of genes in OFT myocardium could lead toaberrant non-cell autonomous signaling and thus decreased celldeath in adjacent mesenchyme.Several groups have now reported the presence and potential

functions of miRNAs in the heart during development and adulthypertrophic responses. Results from our work and that of otherssuggest that, despite the presence of numerous miRNAs, manyfundamental building blocks of cardiacmorphogenesis such as thenumber of chambers and initial formation of component layers arenot severely affected. However, some of the complex morphoge-netic events that allow for correct formation of the mammalianheart’s detailed architecture are indeed flawed in the absence ofmiRNAs. At least in the heart, then, there is a compelling analogybetween the fine-tuning of gene expression thatmiRNAs are oftensuggested to regulate and the resultant fine detailing of cardiacmorphogenesis that is necessary for 4-chambered, dual-outflowcardiac functionality in mammals. As tools to study miRNAscontinue evolving and become more powerful, new details willsurely emerge regarding miRNA-based regulation of cardiac de-velopment and of embryogenesis as a whole.

MethodsGeneration of Dicer Conditional Mice. Nkx2.5-Cre mice (9) and Dicerflox/flox

mice (11) have been previously described and were intercrossed to produceNkx2.5-Cre; Dicerflox/flox mice that were genotyped as described. Nkx2.5-CreCre/+;Dicerflox/flox (mutant) embryos were compared in all analyses toNkx2.5-Cre+/+;Dicerflox/Dicerflox (wild-type) littermates.

RNA In Situ Hybridization. Section in situ hybridization was done with anti-sense riboprobes as previously described (34, 35). RNA expression comparedbetween wild-type and mutant littermates included that of the followinggenes known to be involved in cardiogenesis: Fgf8, GATA4, Hand2, Mef2c,Mlc2v, Pitx2c, PlexinA2, Sema3c, Tbx1.

Immunohistochemistry and TUNEL. Some embryos were embedded in paraffinafter fixation in 4% paraformaldehyde whereas others were fixed with 4%paraformaldehyde followed by 10% and 30% sucrose gradients for cry-osectioning. Serial paraffin-embedded sections were used for H&E staining,RNA in situ hybridization, HHF35 staining, and TUNEL staining; cryosectionswere used for H3B staining and TUNEL staining. Apoptosis was assayed usingProMega’s DeadEnd Fluorometric TUNEL System (#G3250), cell proliferationwith α-phosphohistone H3B (Upstate Biotechnology, # 06–570) and muscleactin with HHF35 (Dako # M0635). Quantitation for apoptosis was done bycounting the number of nonmyocardial apoptotic cells divided by the totalnumber of DAPI-stained nonmyocardial cells per high magnification field ofview with four levels assayed for each embryo. Seven wild-type and sevenmutant embryos were assayed. IMARIS software (Bitplane) was used to assistin identification and counting of nonmyocardial cells.

Statistical Analysis. Statistical calculations were performed using t test ofvariables (two-sample t test assuming unequal variances) with 95% con-fidence intervals.

ACKNOWLEDGMENTS. The authors thank Dane Loeliger for technicalassistance, Dr. Richard Harvey for Nkx2.5-Cre mice, and Dr. Jonathan Epsteinfor plasmids. This work was supported by National Institutes of HealthGrant HD047360.

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