Developmental Biology 362 (2012) 50–56

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Developmental Biology

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Dgcr8 controls neural crest cells survival in cardiovascular development

Elik Chapnik a, Vered Sasson a, Robert Blelloch b, Eran Hornstein a,⁎a Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, 76100, Israelb The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, and Department of Urology, University of CaliforniaSan Francisco, San Francisco, CA 94143, USA

⁎ Corresponding author at: Department of MolecularWeizmann Institute of Science, PO Box 26, Rehovot 7610

E-mail address: eran.hornstein@weizmann.ac.il (E. H

0012-1606/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.ydbio.2011.11.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received for publication 9 October 2011Accepted 12 November 2011Available online 23 November 2011

Keywords:Dgcr8DiGeorgemiRNAmicroRNACardiac neural crestOutflow tract

DiGeorge syndrome (DGS), characterized genetically by a deletion within chromosome 22q11.2, is associatedwith a constellation of congenital heart defects. DiGeorge critical region 8 (Dgcr8), a gene that maps to thecommon deletion region of DGS, encodes a double stranded RNA-binding protein that is essential formiRNA biogenesis. To address the potential contribution of Dgcr8 insufficiency to cardiovascular develop-ment, we have inactivated Dgcr8 in cardiac neural crest cells (cNCCs). Dgcr8 mutants displayed a wide spec-trum of malformations, including persistent truncus arteriosus (PTA) and ventricular septal defect (VSD).Interestingly, Dgcr8-null cNCCs that properly migrated into the cardiac outflow tract (OFT), proliferate nor-mally and differentiate into vascular smooth muscle cells. However, loss of Dgcr8 causes a significant portionof the cNCCs to undergo apoptosis, causing a decrease in the pool of progenitors required for OFT remodeling.Our data uncover a new role of Dgcr8 in cardiovascular morphogenesis, plausibly as part of transmissionmechanism for FGF-dependent survival cue for migrating cNCCs.

© 2011 Elsevier Inc. All rights reserved.

Introduction

The genetic etiology of DiGeorge syndrome (DGS) involves a3 Mbp microdeletion of the DiGeorge critical region (DGCR) onhuman chromosome 22q11.2, thus it is also known as del22q11.2syndrome. Cardiovascular defects observed in DGS, such as inter-rupted aortic arch and ventricular septal defect (VSD) are also ob-served in several mouse experimental models, in which theexpression of genes annotated to the microdeletion 22q11.2 was per-turbed (Lindsay, 2001). However, recapitulation of the full-blownhuman phenotype, including persistent truncus arteriosus (PTA) re-quires homozygous deletion of 22q11.2 genes, such as Tbx1, suggest-ing that other genes potentially contribute to DGS pathogenesis.

Indeed, many other mouse models provided important genetic in-sights into the development of the cardiovascular system and its asso-ciated diseases, including Crkl, TGFbr2, ERK1/2 and Shp2 (Guris et al.,2001; Nakamura et al., 2009; Newbern et al., 2008; Wurdak et al.,2005). Many of these experimental models focus on the cardiac neu-ral crest cells (cNCCs), a unique subset of cells that migrate from thedorsal aspect of the neural tube to remodel the pharyngeal arch arter-ies (PAAs) and the septation of the cardiac outflow tract (OFT) intotwo individual vessels: the pulmonary trunk and ascending aorta(Hutson and Kirby, 2007). Thus, neural crest (NC)-specific knockoutmodels have proved useful in unraveling facets of the molecular net-work governing OFT development and in addition provided tools to

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study genes that hold different dosage threshold between themouse and the human cardiovascular systems (High et al., 2007;Lepore et al., 2006; Vallejo-Illarramendi et al., 2009).

microRNAs (miRNAs) have emerged as central regulators of NC andcardiovascular development (Cordes et al., 2009; Small and Olson,2011; Xin et al., 2009). The maturation of most miRNAs is governedby themicroprocessor complex, composed of the RNase III-like enzymeDROSHA and the RNA-binding protein, DGCR8 (Seitz and Zamore,2006). As suggested by its name, Dgcr8 is mapped within the 22q11.2DGCR locus (and the syntenic region onmouse chromosome 16), mak-ing it a potentially important component in the pathogenesis of DGS(Shiohama et al., 2003). Furthermore, disruption of Dgcr8 results in be-havioral and neuronal deficits characteristic of DGS (Schofield et al.,2011; Stark et al., 2008). Therefore, we sought to analyze the potentialrole of Dgcr8 in cardiovascular development. Here, we describe con-genital heart abnormalities that result from genetic inactivation of aDgcr8 conditional allele in neural crest cells (NCCs).

Materials and methods

Mouse genetics

Dgcr8 conditional allele (Wang et al., 2007) was crossed to a trans-genic Cre line under the control of the Wnt1 promoter (a gift fromAndrew McMahon). To generate Dgcr8 conditional knockout embry-os, Wnt1-cre;Dgcr8loxp/+ males were crossed with Dgcr8loxp/loxp fe-males. R26R-lacZ or R26R-YFP reporter lines, generously shared byP. Soriano and F. Constantini, respectively, were crossed into theDgcr8loxp/loxp females for genetic fate tracing.

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Intracardial ink injection

India ink was injected into the embryo left ventricle using a pulledcapillary tube. Injected embryos were subsequently fixed in 4% PFAovernight, dehydrated by graded methanol washes and cleared inbenzyl benzoate: benzyl alcohol (1:1).

Histology, immunochemistry and proliferation assays

Embryos were collected at stages of interest, rinsed in PBS, fixedovernight in 4% buffered paraformaldehyde at 4 °C, dehydrated througha series of ethanol baths and embedded in Paraplast Plus (McCormickScientific). Paraffin sectionswere subjected to hematoxylin-Eosin stain-ing or to immunofluorescent analysis: Following rehydration, slideswere incubated in 10 mmol/L sodium citrate buffer pH6.0 on a 2100 Re-triever (PickCell Laboratories) for a period of 3 h. Slides were thenwashed in DDW, PBS and 0.1% Tween (Sigma) and blocked with CAS-Block (Invitrogen) for 10 min at room temperature. Primary antibodiesin CAS-Block were incubated overnight at 4 °C: DGCR8 (10996-1-AP,1:100 dilution, Proteintech), GFP (ab6658, 1:300, Abcam), Smoothmuscle α-actin (A 2547, 1:400, DAKO), cleaved Caspase-3 (9661,1:300, Cell Signaling Technology). For proliferation assay, 1 h after in-jection of BrdU at 100 μg/g body weight (Sigma), embryos were har-vested and fixed in 4% paraformaldehyde. Detection of BrdU-labeledcells was performed by using an anti-BrdU antibody (RPN202, 1:300,GE Healthcare). Cyanine conjugated anti-rabbit, anti-mouse or anti-goat secondary antibodies (1:100, ImmunoResearch Laboratories)were added in CAS-Block for 1 h at room temperature. 4′,6-Diami-dino-2-phenylindole dihydrochloride (DAPI) was added for 5 min be-fore slides were mounted with Immu-mount (Thermo Scientific).

A

C D E

B

C’ D’ E’

Fig. 1. Severe cardiovascular malformations observed in Dgcr8 mutants. Sagittal aortic arDgcr8loxp/loxp;R26R-YFP mutants (B, n=4) stained for YFP (green) and DGCR8 (red). MagWnt1-cre;Dgcr8loxp/loxp mutants (D–E, n=11) and respective schemes of the great vesselsn=11/11), while additional abnormalities included interrupted aortic arch type B (IAA; E′vical aortic arch (Cx-AA; D′ n=2/11). RSA, right subclavian artery; RCC, right common carotarch; Ao, aorta; DA, ductus arteriosus. Anterior (top) view of E18.5 OFT, divided into pulmonWnt1-cre;Dgcr8loxp/loxp (G). H&E staining of representative E18.5 frontal heart sections fromwith persistent truncus arteriosus (PTA) and ventricular septal defect (VSD). Ao, aorta; PAthymus (J, n=4) and Wnt1-cre;Dgcr8loxp/loxp thymus that is malformed and ectopically poslobe; Tr, trachea.

Fluorescent images were captured with a Nikon 90i mounted withNikon D5-SM camera or a Zeiss LSM510 Laser Scanning confocal mi-croscopy system.

Whole mount RNA in situ hybridization

Embryos were harvested and fixed in 4% paraformaldehyde over-night at 4 °C and dehydrated in gradedmethanols. DIG-labeled antisenseRNA probes for Crabp1 and Ap2-alpha were transcribed in vitro (RocheApplied Science) following manufacturer's instructions.

Whole mount β-galactosidase staining

Embryos were fixed in fresh 4% paraformaldehyde at 4 °C for30 min, thoroughly washed with PBS, supplemented with 0.02% NP-40 (Sigma) and developed in 20 mmol/L potassium ferricyanide,20 mmol/L potassium ferrocyanide, 2 mmol/L magnesium chlorideand 0.4 mg/mL X-gal substrate in PBS, 0.02% NP-40, at 37 °C for 4 h.Some embryos were further paraffin embedded and sectioned.

Results

Deletion of Dgcr8 in NCCs leads to severe cardiovascular defects

cNCCs remodel the PAAs and enable septation of the cardiac OFTinto the pulmonary artery and the aorta (Hutson and Kirby, 2007).To assess Dgcr8 requirement in PAAs and OFT development, we gen-eratedWnt1-cre;Dgcr8loxp/loxp mice by crossing a Dgcr8 conditional al-lele (Wang et al., 2007) onto a Cre transgene that is driven by theWnt1 promoter. NC-specific Cre activity resulted in loss of DGCR8

F G

H I

J K

ch sections of E10.5 Wnt1-cre;Dgcr8loxp/+;R26R-YFP controls (A, n=3) and Wnt1-cre;nified view in insets. (C–E) Gross thorax anatomy of E17.5 controls (C, n=10) and(C′–E′). All Wnt1-cre;Dgcr8loxp/loxp displayed persistent truncus arteriosus (PTA; D′,E′n=3/11), aberrant origin of the right subclavian artery (Ab-RSA; D′ n=3/11) and cer-id; IA, inominate artery; LSA, left subclavian artery; LCC, left common carotid; AA, aorticary artery and aorta in healthy controls (F) and displays persistent truncus arteriosus incontrols (H, n=3) and Wnt1-cre;Dgcr8loxp/loxp mutants (I, n=3), the latter manifests

, pulmonary artery; RV, right ventricle; LV, left ventricle. Frontal view of E18.5 controlitioned, in a lateral situs in the thorax (K, n=3). LT, left thymic lobe; RT, right thymic

52 E. Chapnik et al. / Developmental Biology 362 (2012) 50–56

and also in induction of a ROSA26 YFP-knock-in reporter transgene(R26R-YFP) (Figs. 1A,B). Since DGCR8 was not detected at embryonicday (E) 10.5 inWnt1-cre;Dgcr8loxp/loxp NC-descendants, that were rec-ognized by activation of the R26R-YFP reporter, we conclude that Cremediated recombination efficiently disrupted the Dgcr8 gene(Figs. 1A,B).

While Wnt1-cre;Dgcr8loxp/+ and Dgcr8loxp/loxp mice were phenotypi-cally normal and taken as controls throughout this study, Wnt1-cre;Dgcr8loxp/loxp littermates died perinatally. Gross anatomical explorationof Wnt1-cre;Dgcr8loxp/loxp mice revealed major cardiovascular defects atE18.5, including PTA (100%), interrupted aortic arch (27%), cervical aor-tic arch (18%) and aberrant origin of the right subclavian artery (27%)(Figs. 1C–G). Coronal sections of dissectedWnt1-cre;Dgcr8loxp/loxp hearts

A B

C D

E F

M

Fig. 2. Normal migration but reduced number of neural crest-descendants in Dgcr8mutant Oand Wnt1-cre;Dgcr8loxp/loxp mutant (B, n=8). 3, 3rd PAA; 4, 4th PAA; 6, 6th PAA. Whole mon=5 per genotype) on E10.5 Wnt1-cre;Dgcr8loxp/loxp embryos (D,F) and control littermatesWhole-mount beta-gal staining of E10.5 embryos, expressing a R26R-LacZ lineage tracingHigher magnification reveals seemingly-normal cNCCs migration into the pharyngeal archblack arrowhead denotes the distal-most LacZ-positive cells in the OFT. Sagittal sections ofryngeal arch and the developing cardiac OFT, of control littermates (K) and Wnt1-cre;Dgcr8sing R26R-LacZ, reveals reduced cNCC numbers at the OFT of Wnt1-cre;Dgcr8loxp/loxp mutantspulmonary artery; PTA, persistent truncus arteriosus.

further revealed a VSD (Figs. 1H,I). In addition we noted hypoplastic,malpositioned thymi (Figs. 1J,K) and craniofacial abnormalities (Fig.S1). This spectrum of defects is reminiscent of human cardiovasculardiseases, including manifestations described in DGS.

Initial pharyngeal arch artery formation is not disrupted in Dgcr8mutants

To study potential early PAA patterning defects, we performed in-tracardial ink injection on E10.5 embryos. The PAAs were well-formed and patent in the majority of Wnt1-cre;Dgcr8loxp/loxp embryos(n=7/8) and in all of their control littermates (n=24/24; Figs. 2A,B).Therefore, ablation of Dgcr8 in cNCCs cells does not disrupt initial PAA

G H

I J

K L

N

FT. India ink-injected E10.5 pharyngeal arch arteries. Lateral view of control (A, n=24)unt in situ hybridization study of Ap2-alpha (C,D, n=5, per genotype), and Crabp1 (E,F,(C,E). Arrows indicate the migratory streams of NCCs entering the pharyngeal arches.reporter in control (G) and Wnt1-cre;Dgcr8loxp/loxp (H) embryos (n=4, per genotype).es and OFT of controls (I) and Wnt1-cre;Dgcr8loxp/loxp (J). PA1, 1st pharyngeal arch. AE10.5 embryos confirm the presence of NC-derived, LacZ-positive cells, in the 1st pha-loxp/loxp mutants (L). Whole-mount beta-gal staining of E12.5 embryonic hearts expres-(N, n=4, denoted by a black arrowhead), relative to controls (M, n=3). Ao, aorta; PA,

A

B

C

D

E

Fig. 3. Dgcr8mutant cNCC descendants exhibit normal proliferation and smooth muscle differentiation. Immunofluorescent analysis of E10.5 sagittal sections reveals co-localizationof α-smooth muscle actin (SMA-α, red) and cNCC genetic fate tracer R26R-YFP (Green) in the OFT of control (A, n=3) andWnt1-cre;Dgcr8loxp/loxp mutant (B, n=3). Yellow, mergedgreen and red signals. Representative cells at the OFT, positive for SMA-α and for R26R-YFP, are denoted by white arrowheads in A and B, respectively. Transverse sections of E10.5embryos at the level of the OFT reveals comparable levels of BrdU incorporation in E10.5 Wnt1-cre;Dgcr8loxp/loxp (D) relative to controls (C). Red, BrdU; Green, YFP; Blue, DAPI. (E)Quantification of the percentage of BrdU-positive cells out of the YFP-positive (cNCCs) in mutants (red) and controls (blue, n=4 per genotype). n.s. not statistically significant.

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formation (Lindsay, 2001). We conclude that aortic arch malforma-tions observed in Wnt1-cre;Dgcr8loxp/loxp embryos develop afterE10.5 and are likely related to the remodeling of the great vessels.

Normal migration of cNCCs into the OFT region

cNCCs migrate in trajectories that pass through PAAs into thecardiac OFT (Hutson and Kirby, 2007). To evaluate cNCC migrationwe studied the expression of Crabp1 and Ap2-α, two NC markers.At E10.5, the expression pattern of these markers was comparablein Wnt1-cre;Dgcr8loxp/loxp and control embryos (Figs. 2C–F), suggest-ing normal NC migration into the PAAs and OFT. We have substanti-ated our analysis of NC migration by genetic fate tracing, usingWnt1-cre;Dgcr8loxp/loxp;R26R-LacZ embryos. At E10.5 we were able todetect LacZ-positive Dgcr8loxp/loxp;Wnt1-cre;R26R-LacZ cNCC-descendantson their path into the PAAs and the cardiac OFT, that were indistinguish-able from controls (Figs. 2G-L). However, two days later, at E12.5, weobserved a significant reduction in LacZ expression of Wnt1-cre;Dgcr8loxp/loxp cNCC-descendants in the developing OFT, relative tocontrols (Figs. 2M,N). cNCCs enter the heart in two parallel prongsthat are stereotypically positioned on opposite sides of the cardiacOFT (Hutson and Kirby, 2007). In Wnt1-cre;Dgcr8loxp/loxp OFT, thetwo prongs were malformed (Figs. 2M,N). Plausibly, perturbed OFTmorphogenesis was due to reduced cell numbers that first manifestsbetween E10.5 and E12.5.

Normal smooth muscle differentiation and proliferation of cNCCs in theabsence of Dgcr8.

Normal septum buildup and conotruncus remodeling requiresthat cNCCs-descendants will differentiate into vascular smooth mus-cle (Hutson and Kirby, 2007; Kirby et al., 1983). To evaluate the im-pact of Dgcr8 ablation on cNCC differentiation into smooth muscle,we immunostained sections of the cardiac OFT with α-smooth mus-cle actin (SMA-α). This analysis revealed normal differentiation ca-pacity of cNCCs into smooth muscle, even in the absence of Dgcr8(Figs. 3A,B).

DGCR8 is a regulator of cell cycle (Wang et al., 2007), therefore wehypothesized that the reduction in cNCC numbers observed in E12.5Wnt1-cre;Dgcr8loxp/loxp OFTs could stem from impaired proliferation.However, 5-bromo-2′-deoxyuridine (BrdU) incorporation analysisrevealed a comparable percentage of cNCC-descendants engaged inmitotic cell cycle at E10.5, E11.5 and E12.5, in both Wnt1-cre;Dgcr8loxp/loxp and controls (Figs. 3C-E and unpublished data). Thus,the apparent decrease in cell numbers observed at E12.5 mutantOFTs, is not due to inferior proliferation.

Inactivation of Dgrc8 in NCCs leads to elevated apoptosis

Loss of Dgcr8 was previously linked to enhanced apoptosis (Yi etal., 2009), another potential reason for the disappearance of cNCCsin the OFTs of E12.5 Wnt1-cre;Dgcr8loxp/loxp. Indeed, E10.5 Wnt1-cre;

A B

C D

E

F G H

I J K

Fig. 4. Dgcr8 mutant cNCC descendants exhibit enhanced cell death and attenuated ERK signaling. Cleaved caspase 3 (cCasp-3) immunofluorescent analysis of E10.5 sagittal pha-ryngeal arch (A,C) or outflow tract (OFT, B,D) sections. PA1-6 denote the 1st to the 6th pharyngeal arches, respectively; (n=3 per genotype). (E) Quantification of cleaved caspase 3(cCasp-3) immunoreactive cells reveals enhanced apoptosis in the pharyngeal arch region ofWnt1-cre;Dgcr8loxp/loxp mutant (red) relative to control (blue), but not in the OFT per-se.* pb0.05. n.s. not statistically significant. Immunofluorescent analysis of E10.5 sagittal pharyngeal arch sections reveals significant downregulation of phosphorylated Erk1/2 inWnt1-cre;Dgcr8loxp/loxp 1st and 2nd pharyngeal arches (I–K, J,K are enlargements of insets in I), relative to controls (F–H, G,H are enlarged micrographs of insets in F. n=3, pergenotype).

54 E. Chapnik et al. / Developmental Biology 362 (2012) 50–56

Dgcr8loxp/loxp exhibited enhanced cleaved Caspase-3 staining in pha-ryngeal arches 1–4 and 6 (Figs. 4C,D), whilst control embryos exhib-ited only limited, physiological apoptosis (Figs. 4A,B). Intriguingly,apoptosis of cNCC descendants was not identified at the OFT region(Figs. 4B,D,E), suggesting rather unexpectedly, that cNCCs cells aresusceptible to apoptosis at some positions along their journey butthat post-migration they can survive without DGCR8 activity. Whenconsidered together, it appears that cNCCs normally migrate intothe OFT region until E10.5 but reduced total cell numbers hampersnormal morphogenesis of the aortic-pulmonary septum. This de-crease in cell numbers is not due to inferior proliferation but becausemany Dgcr8-null cNCCs undergo apoptosis at the caudal pharyngealarches, just before approaching the OFT region.

ERK signaling is attenuated in Dgcr8-null pharyngeal arches

In the pharyngeal arches, secreted FGF acts as a survival signal formigratory cNCCs through activation of an intracellular ERK cascade. Dis-ruption of FGF/ERK signaling gives rise to cardiovascular malformationsthat are associated with DGS and are reminiscent of those observed inWnt1-cre;Dgcr8loxp/loxp mice (Frank et al., 2002; Krenz et al., 2008;Newbern et al., 2008). Furthermore, loss ofmiRNA activity downstream

of Dicer attenuates ERK signaling in NCCs (Huang et al., 2010). There-fore, we examined the expression level of phosphorylated Erk1/2 pro-teins in Wnt1-cre;Dgcr8loxp/loxp embryos. Our confocal analysisrevealed significantly decreased expression of phosphorylated Erk1/2in E10.5Wnt1-cre;Dgcr8loxp/loxp pharyngeal arches (Figs. 4F–K), reminis-cent of theDicer1-null phenotype and suggesting it as a likely contribut-ing factor for apoptosis (Figs. 4F–K).

Taken together, our data provide in vivo evidence for Dgcr8 func-tion in cardiovascular development and potentially associates Dgcr8,which is positioned in humans on chromosome 22q11.2, to DGS.

Discussion

Dgcr8 is annotated to the 22q11.2 microdeletion (Shiohama et al.,2003) and Dgcr8 heterozygous disruption results in behavioral andneuronal deficits characteristic of DGS (Schofield et al., 2011; Starket al., 2008). In DGS, cardiovascular structures are frequently affected,as a result of imperfect morphogenesis in embryonic stages (Ryan etal., 1997), related to the contribution of cNCCs. In this work we usedmouse genetics to study how loss of Dgcr8 in NCCs impinges on car-diovascular and pharyngeal development.

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Our data suggest that Dgcr8 is essential for the normal patterningof the OFT, since Wnt1-cre;Dgcr8loxp/loxp embryos develop with car-diovascular defects reminiscent of those reported in humans, in-cluding in DGS patients. Accordingly, Dgcr8 mutants fail toremodel the conotruncus into independent aorta and pulmonary ar-tery and exhibit VSD, interrupted aortic arch, and aberrant origin ofthe right subclavian artery.

Genetic fate mapping revealed normal early cNCCs migration intothe OFT region at E10.5. However, many Dgcr8-null cNCCs undergoapoptosis at the caudal pharyngeal arches, just before approachingthe OFT region. Thus, reduced total cell number hampers normal mor-phogenesis of the aortic-pulmonary septum and the ventricularseptum.

FGF/ERK signaling provides essential survival cues for migratoryNCCs. Furthermore, apoptosis was reported in migratory cNCCs ofFGF hypomorphs, or when ERK was conditionally inactivated inNCCs (Frank et al., 2002; Krenz et al., 2008; Newbern et al., 2008).We therefore suggest that the necessity for Dgcr8-dependant FGF/ERK survival cues is restricted to the pharyngeal region. Intriguingly,cells that manage to inhabit the OFT, are protected from apoptosis,even if Dgcr8 ceases to be expressed. This further supports the roleof FGF signaling in the Wnt1-cre;Dgcr8loxp/loxp model, since the fieldof FGF activity is defined within the pharyngeal arches and does notextend into the OFT, as revealed by the expression domains of FGFdownstream effectors, Pea3 and ERM (Chotteau-Lelievre et al.,2001; Mesbah et al., 2008).

Dgcr8 effectors are very likely miRNA genes (Gregory et al., 2004;Han et al., 2004). miRNA are involved in NCC migration, pharyngealarch development and smooth muscle differentiation (Cordes et al.,2009; Small and Olson, 2011; Xin et al., 2009). For example, miR-452 was recently shown to mediate non-cell-autonomous molecularcrosstalk between the Shh and FGF signaling pathways in the firstpharyngeal arch (Sheehy et al., 2010). Similarly, miR-145 controlsNCC differentiation into smooth muscle upstream of SRF and Myocar-din (Cordes et al., 2009). These specific miRNAs may be intriguing ef-fectors of DGCR8, both in mouse models and in human pathology.

Interestingly, conditional inactivation of Dicer1 in NCCs by aWnt1-cre deleter caused OFT abnormalities that closely resemblethe defects reported here (Huang et al., 2010; Sheehy et al., 2010).In addition, ERK signaling is attenuated in the Wnt1-cre;Dicer1model, (Huang et al., 2010) suggesting that the regulation of FGF sig-naling is likely mediated, intra-cellularly, by miRNAs in both mousemodels. These results strongly suggest that Dgcr8may work upstreamof Dicer1 in cNCC development. Nonetheless, and quite surprisingly,in the Wnt1-cre;Dicer1 model, apoptosis was limited to the first pha-ryngeal arch but was not detected in the caudal pharyngeal arches orthe OFT (Huang et al., 2010). This difference maybe molecularly relat-ed to DICER1 activity in other pathways including: siRNA, heterochro-matin, and functional silencing of toxic Alu transcripts, (Kaneko et al.,2011; Tam et al., 2008; Watanabe et al., 2008) which are independentof DGCR8 and miRNAs.

In summary, our study characterizes the essential role of Dgcr8, aChr22q11.2 gene, in OFT morphogenesis and should motivate re-search of mutations in the Dgcr8 gene or specific miRNA genes inhumans that manifest with cardiovascular defects.

Supplementary materials related to this article can be found on-line at doi:10.1016/j.ydbio.2011.11.008.

Acknowledgments

We thank Andy McMahon for the Wnt1-cre allele, Phil Sorianoand Frank Constantini for Rosa26 reporter lines. This work was sup-ported by grants to EH from the Israel Science Foundation, the estatesof F. Blau; L. Asseof; F. Sherr; C. Vener; J. Benattar; D. Francouer(Merck); Lilly Fulop; N. Baltor and M. Halphen, Yeda-Sela Center forBasic Research, and the Wolfson Family Charitable Trust. EH is the

incumbent of the Dr. Sydney Brenner Chair and the Helen and MiltonA Kimmelman Career Development Chair.

References

Chotteau-Lelievre, A., Dolle, P., Peronne, V., Coutte, L., de Launoit, Y., Desbiens, X., 2001.Expression patterns of the Ets transcription factors from the PEA3 group duringearly stages of mouse development. Mech. Dev. 108, 191–195.

Cordes, K.R., Sheehy, N.T., White, M.P., Berry, E.C., Morton, S.U., Muth, A.N., Lee, T.H.,Miano, J.M., Ivey, K.N., Srivastava, D., 2009. miR-145 and miR-143 regulate smoothmuscle cell fate and plasticity. Nature 460, 705–710.

Frank, D.U., Fotheringham, L.K., Brewer, J.A., Muglia, L.J., Tristani-Firouzi, M., Capecchi,M.R., Moon, A.M., 2002. An Fgf8 mouse mutant phenocopies human 22q11 deletionsyndrome. Development 129, 4591–4603.

Gregory, R.I., Yan, K.P., Amuthan, G., Chendrimada, T., Doratotaj, B., Cooch, N., Shiekhattar,R., 2004. The microprocessor complex mediates the genesis of microRNAs. Nature432, 235–240.

Guris, D.L., Fantes, J., Tara, D., Druker, B.J., Imamoto, A., 2001. Mice lacking the homo-logue of the human 22q11.2 gene CRKL phenocopy neurocristopathies of DiGeorgesyndrome. Nat. Genet. 27, 293–298.

Han, J., Lee, Y., Yeom, K.H., Kim, Y.K., Jin, H., Kim, V.N., 2004. The Drosha-DGCR8 com-plex in primary microRNA processing. Genes Dev. 18, 3016–3027.

High, F.A., Zhang, M., Proweller, A., Tu, L., Parmacek, M.S., Pear, W.S., Epstein, J.A., 2007.An essential role for Notch in neural crest during cardiovascular development andsmooth muscle differentiation. J. Clin. Invest. 117, 353–363.

Huang, Z.P., Chen, J.F., Regan, J.N., Maguire, C.T., Tang, R.H., Rong Dong, X., Majesky, M.W.,Wang, D.Z., 2010. Loss of MicroRNAs in neural crest leads to cardiovascular syn-dromes resembling human congenital heart defects. Arterioscler. Thromb. Vasc. Biol.30, 2575–2586.

Hutson, M.R., Kirby, M.L., 2007. Model systems for the study of heart development anddisease. Cardiac neural crest and conotruncal malformations. Semin. Cell Dev. Biol.18, 101–110.

Kaneko, H., Dridi, S., Tarallo, V., Gelfand, B.D., Fowler, B.J., Cho, W.G., Kleinman, M.E.,Ponicsan, S.L., Hauswirth, W.W., Chiodo, V.A., Kariko, K., Yoo, J.W., Lee, D.K.,Hadziahmetovic, M., Song, Y., Misra, S., Chaudhuri, G., Buaas, F.W., Braun, R.E.,Hinton, D.R., Zhang, Q., Grossniklaus, H.E., Provis, J.M., Madigan, M.C., Milam,A.H., Justice, N.L., Albuquerque, R.J., Blandford, A.D., Bogdanovich, S., Hirano,Y., Witta, J., Fuchs, E., Littman, D.R., Ambati, B.K., Rudin, C.M., Chong, M.M., Provost,P., Kugel, J.F., Goodrich, J.A., Dunaief, J.L., Baffi, J.Z., Ambati, J., 2011. DICER1 def-icit induces Alu RNA toxicity in age-related macular degeneration. Nature 471,325–330.

Kirby, M.L., Gale, T.F., Stewart, D.E., 1983. Neural crest cells contribute to normal aorti-copulmonary septation. Science 220, 1059–1061.

Krenz, M., Gulick, J., Osinska, H.E., Colbert, M.C., Molkentin, J.D., Robbins, J., 2008. Roleof ERK1/2 signaling in congenital valve malformations in Noonan syndrome. Proc.Natl. Acad. Sci. U. S. A. 105, 18930–18935.

Lepore, J.J., Mericko, P.A., Cheng, L., Lu, M.M., Morrisey, E.E., Parmacek, M.S., 2006.GATA-6 regulates semaphorin 3C and is required in cardiac neural crest for cardio-vascular morphogenesis. J. Clin. Invest. 116, 929–939.

Lindsay, E.A., 2001. Chromosomal microdeletions: dissecting del22q11 syndrome. Nat.Rev. Genet. 2, 858–868.

Mesbah, K., Harrelson, Z., Theveniau-Ruissy, M., Papaioannou, V.E., Kelly, R.G., 2008.Tbx3 is required for outflow tract development. Circ. Res. 103, 743–750.

Nakamura, T., Gulick, J., Colbert, M.C., Robbins, J., 2009. Protein tyrosine phosphataseactivity in the neural crest is essential for normal heart and skull development.Proc. Natl. Acad. Sci. U. S. A. 106, 11270–11275.

Newbern, J., Zhong, J., Wickramasinghe, R.S., Li, X., Wu, Y., Samuels, I., Cherosky, N.,Karlo, J.C., O'Loughlin, B., Wikenheiser, J., Gargesha, M., Doughman, Y.Q., Charron,J., Ginty, D.D., Watanabe, M., Saitta, S.C., Snider, W.D., Landreth, G.E., 2008. Mouseand human phenotypes indicate a critical conserved role for ERK2 signaling inneural crest development. Proc. Natl. Acad. Sci. U. S. A. 105, 17115–17120.

Ryan, A.K., Goodship, J.A., Wilson, D.I., Philip, N., Levy, A., Seidel, H., Schuffenhauer, S.,Oechsler, H., Belohradsky, B., Prieur, M., Aurias, A., Raymond, F.L., Clayton-Smith,J., Hatchwell, E., McKeown, C., Beemer, F.A., Dallapiccola, B., Novelli, G., Hurst,J.A., Ignatius, J., Green, A.J., Winter, R.M., Brueton, L., Brondum-Nielsen, K., Scambler,P.J., et al., 1997. Spectrum of clinical features associated with interstitial chromosome22q11 deletions: a European collaborative study. J. Med. Genet. 34, 798–804.

Schofield, C.M., Hsu, R., Barker, A.J., Gertz, C.C., Blelloch, R., Ullian, E.M., 2011. Monoalle-lic deletion of the microRNA biogenesis gene Dgcr8 produces deficits in the devel-opment of excitatory synaptic transmission in the prefrontal cortex. Neural Dev.6, 11.

Seitz, H., Zamore, P.D., 2006. Rethinking the microprocessor. Cell 125, 827–829.Sheehy, N.T., Cordes, K.R., White, M.P., Ivey, K.N., Srivastava, D., 2010. The neural crest-

enriched microRNA miR-452 regulates epithelial-mesenchymal signaling in thefirst pharyngeal arch. Development 137, 4307–4316.

Shiohama, A., Sasaki, T., Noda, S., Minoshima, S., Shimizu, N., 2003. Molecular cloningand expression analysis of a novel gene DGCR8 located in the DiGeorge syndromechromosomal region. Biochem. Biophys. Res. Commun. 304, 184–190.

Small, E.M., Olson, E.N., 2011. Pervasive roles of microRNAs in cardiovascular biology.Nature 469, 336–342.

Stark, K.L., Xu, B., Bagchi, A., Lai, W.S., Liu, H., Hsu, R., Wan, X., Pavlidis, P., Mills, A.A.,Karayiorgou, M., Gogos, J.A., 2008. Altered brain microRNA biogenesis contrib-utes to phenotypic deficits in a 22q11-deletion mouse model. Nat. Genet. 40,751–760.

56 E. Chapnik et al. / Developmental Biology 362 (2012) 50–56

Tam, O.H., Aravin, A.A., Stein, P., Girard, A., Murchison, E.P., Cheloufi, S., Hodges, E.,Anger, M., Sachidanandam, R., Schultz, R.M., Hannon, G.J., 2008. Pseudogene-derivedsmall interfering RNAs regulate gene expression in mouse oocytes. Nature 453,534–538.

Vallejo-Illarramendi, A., Zang, K., Reichardt, L.F., 2009. Focal adhesion kinase is requiredfor neural crest cell morphogenesis during mouse cardiovascular development.J. Clin. Invest. 119, 2218–2230.

Wang, Y., Medvid, R., Melton, C., Jaenisch, R., Blelloch, R., 2007. DGCR8 is essential formicroRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat.Genet. 39, 380–385.

Watanabe, T., Totoki, Y., Toyoda, A., Kaneda, M., Kuramochi-Miyagawa, S., Obata, Y.,Chiba, H., Kohara, Y., Kono, T., Nakano, T., Surani, M.A., Sakaki, Y., Sasaki, H.,

2008. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts inmouse oocytes. Nature 453, 539–543.

Wurdak, H., Ittner, L.M., Lang, K.S., Leveen, P., Suter, U., Fischer, J.A., Karlsson, S., Born, W.,Sommer, L., 2005. Inactivation of TGFbeta signaling in neural crest stem cells leads tomultiple defects reminiscent of DiGeorge syndrome. Genes Dev. 19, 530–535.

Xin, M., Small, E.M., Sutherland, L.B., Qi, X., McAnally, J., Plato, C.F., Richardson, J.A.,Bassel-Duby, R., Olson, E.N., 2009. MicroRNAs miR-143 and miR-145 modulatecytoskeletal dynamics and responsiveness of smooth muscle cells to injury. GenesDev. 23, 2166–2178.

Yi, R., Pasolli, H.A., Landthaler, M., Hafner, M., Ojo, T., Sheridan, R., Sander, C., O'Carroll,D., Stoffel, M., Tuschl, T., Fuchs, E., 2009. DGCR8-dependent microRNA biogenesis isessential for skin development. Proc. Natl. Acad. Sci. U. S. A. 106, 498–502.