Protein tyrosine phosphatase activity in the neuralcrest is essential for normal heart andskull developmentTomoki Nakamura, James Gulick, Melissa C. Colbert, and Jeffrey Robbins1

Department of Pediatrics, Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229

Edited by Jonathan G. Seidman, Harvard Medical School, Boston, MA, and approved May 6, 2009 (received for review February 27, 2009)

Mutations within the protein tyrosine phosphatase, SHP2, which isencoded by PTPN11, cause a significant proportion of Noonansyndrome (NS) cases, typically presenting with both cardiac diseaseand craniofacial abnormalities. Neural crest cells (NCCs) participatein both heart and skull formation, but the role of SHP2 signaling inNCC has not yet been determined. To gain insight into the role ofSHP2 in NCC function, we ablated PTPN11 specifically in premigra-tory NCCs. SHP2-deficient NCCs initially exhibited normal migra-tory and proliferative patterns, but in the developing heart failedto migrate into the developing outflow tract. The embryos dis-played persistent truncus arteriosus and abnormalities of the greatvessels. The craniofacial deficits were even more pronounced, withlarge portions of the face and cranium affected, including themandible and frontal and nasal bones. The data show that SHP2activity in the NCC is essential for normal migration and differen-tiation into the diverse lineages found in the heart and skull anddemonstrate the importance of NCC-based normal SHP2 activity inboth heart and skull development, providing insight into thesyndromic presentation characteristic of NS.

Approximately 4% of human infants are born with a con-genital malformation. Abnormal heart formation, the most

common human birth defect, aff licts nearly 1% of newborns (1).Numerous clinical syndromes result from mutations that affectregulation of the ERK1/2 cascade (2), including Noonan(PTPN11, K-RAS, C-RAF, SOS1), LEOPARD (PTPN11, NF1,C-RAF), Costello (H-RAS), and cardio-facio-cutaneous (B-RAF, K-RAS, MEK1, MEK2) syndromes (3). Affected patientspresent with several overlapping phenotypes in the brain, heart,face, and skin; thus, these syndromes are termed neurocardio-facial cutaneous (NCFC) syndromes (4). Mutations associatedwith these syndromes often result in gain of function with respectto signaling through ERK1/2, with the exception of certainLEOPARD syndrome (LS) mutations in PTPN11 associatedwith loss of function (5, 6).

PTPN11, located on human chromosome 12q24.1, encodes theubiquitously expressed protein tyrosine phosphatase SHP2.SHP2 is essential for early vertebrate development and canregulate cell migration, proliferation, survival, and differentia-tion through pathways, including the MAPK, ERK1/2 branch(7). Mice homozygous for a null mutation in PTPN11 die atimplantation due to failure in the development of the extraem-bryonic trophoectodermal lineage, whereas induction of a dom-inant-negative SHP2 in Xenopus blocks mesoderm formation byimpairing ERK signaling, leading to arrest of gastrulation (8, 9).

Neural crest cells (NCCs), often referred to as the fourth germlayer, are a multipotent cell population originating after gastru-lation. NCCs are first localized in the neural folds at the dorsalaspect of the developing spinal cord and delaminate from theneural tube. They then migrate via different pathways towardthe organ primordia, where they give rise to and influence thedevelopment of a diverse array of tissues, including numerouscraniofacial structures, the cardiac outflow tract (OFT), theendocrine glands, and neurons (10). Therefore, some of thedefects observed in NCFC syndromes may arise principally from

perturbation of NCC determination, proliferation, migration, ordifferentiation. These NCC processes are regulated by variousmolecular signals and pathways, including those dependent onsonic hedgehog, the Wingless/INT-related families (Wnts), andbone morphogenic proteins and fibroblast growth factors(FGFs) (11). To the best of our knowledge, however, the role ofSHP2 in NCC function has not yet been elucidated. In thepresent work, we used Cre-mediated excision of PTPN11 inpremigratory NCCs to explore the role of SHP2 in this cellpopulation as it participates in the development of the heart andhead fields. The data demonstrate that loss of SHP2 function inthe premigratory neural crest can lead directly to heart and facialdefects, and that this lost function can be largely rescued throughthe restoration of normal ERK1/2 signaling. These data providea mechanistic basis for understanding the syndromic presenta-tions that can occur in such diseases as Noonan syndrome andDiGeorge syndrome as a result of altered signaling in the neuralcrest.

ResultsGeneration of Mice Containing a Conditional Null Mutation of SHP2.SHP2 contains 2 src homology (SH2) domains and a proteintyrosine phosphatase domain (PTP), with the amino terminus-SH2 domain functioning as a molecular switch to regulateSHP2’s catalytic activity (12). To conditionally inactivate theN-SH2 domain and the hinge region linking the 2 SH2 domains,a mutant allele was created by introducing 2 loxP sites intointrons outside exon 3 and 4 [supporting information (SI) Fig.S1A and B]. Genotyping of the homozygous SHP2 floxed mice(SHP2loxP/loxP) via PCR showed the expected fragment of 320bases corresponding to the floxed allele (Fig. S1C). Beforeexcision of the sequences flanked by the loxP sequence, thesemice were viable, fertile, and generally indistinguishable fromcontrol littermates, demonstrating that the conditional gene-targeting strategy did not disrupt normal SHP2 expression. Todetermine the function of SHP2 in NCCs, SHP2 conditionalmutants (SHP2loxP/wt) were interbred to transgenic mice express-ing Cre recombinase under the transcriptional control of theNCC-restricted Wnt1 promoter. The Wnt1 promoter is tran-scriptionally active only in the premigratory NCCs residing in thedorsal neural tube, and Wnt1 expression is extinguished as NCCsmigrate away from the neural tube, with the promoter remainingsilent thereafter (13). Heterozygous SHP2 knockout mice(Wnt1Cre::SHP2�3�4/wt) were completely normal and indistin-guishable from control littermates. Wnt1Cre::SHP2�3–4/wt off-

Author contributions: T.N., J.G., and J.R. designed research; T.N., J.G., and J.R. performedresearch; T.N., J.G., and M.C.C. contributed new reagents/analytic tools; T.N., J.G., and J.R.analyzed data; and T.N. and J.R. wrote the paper.

The authors declare no conflicts of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: jeff.robbins@cchmc.org.

This article contains supporting information online at www.pnas.org/cgi/content/full/0902230106/DCSupplemental.

11270–11275 � PNAS � July 7, 2009 � vol. 106 � no. 27 www.pnas.org�cgi�doi�10.1073�pnas.0902230106

Dow

nloa

ded

by g

uest

on

June

1, 2

020

spring were then interbred with SHP2loxP/loxP mice to generatehomozygous SHP2 knockout mice (Wnt1Cre::SHP2�3–4/�3–4)and control littermates. To determine the efficiency of Cre-mediated excision of PTPN11 in NCCs, SHP2 expression wasassessed using E9.5 pharyngeal arch samples with abundantNCCs. On immunostaining, SHP2 was ubiquitously expressed inthe pharyngeal arches of the control embryos (Fig. S1D), asexpected. These mice were subsequently bred into our CAG-CAT EGFP background, to fate map the NCCs (13); as ex-pected, the EGFP-labeled NCCs showed reduced SHP2 expres-sion in the Wnt1Cre::SHP2�3–4/wt and Wnt1Cre::SHP2�3–4/�3–4

embryos (Fig. S1D). Quantitation by Western blot analysisrevealed SHP2 reductions of 50% in the Wnt1Cre::SHP2�3–4/wt em-bryos and 90% in the Wnt1Cre::SHP2�3–4/�3–4 embryos (Fig. S1E),with the residual 10% of SHP2 in the Wnt1Cre::SHP2�3–4/�3–4

embryos due to its expression in pharyngeal ectoderm andendoderm (Fig. S1D).

Both the Wnt1Cre::SHP2�3–4/wt and Wnt1Cre::SHP2�3–4/�3–4

embryos survived in the anticipated Mendelian ratios until mid-gestation; however, the survival rate for Wnt1Cre::SHP2�3–4/�3–4

embryos dropped after E15.5, and no pups were born alive (TableS1). By E15.5, the Wnt1Cre::SHP2�3–4/�3–4 embryos invariablywere significantly smaller and displayed a characteristic spectrum ofcardiac and craniofacial defects, defined below.

NCC-SHP2 Null Embryos Exhibit Severe Craniofacial Anomalies andHeart Defects. Patients suffering from NS often display visiblecraniofacial phenotypes, including positional anomalies of theeyes and ears; a short, webbed neck; smaller-than-normal jaw;altered canthal distances; down-slanting palpebral fissures; de-pressed nasal root; wide-based nose with a bulbous nasal tip;pointed chin; and a large cranium with a small face. CranialNCCs contribute significantly to the formation of mesenchymalstructures in the head and neck by differentiating into manycell types, including neurons, glia, cartilage, bone, and connectivetissue (14). Compared with controls, the Wnt1Cre::SHP2�3–4/�3–4

embryos had a smaller head, nose, jaw, and ear, as well as eyeplacement anomalies (Fig. 1A). Histological examination of theWnt1Cre::SHP2�3–4/�3–4 embryos revealed defects in the nasophar-ynx, tongue, mandible, and nasal capsule and bone (Fig. 1B).

The 3-dimensional architecture of the skeleton was examinedusing modified whole-mount alcian blue–alizarin red S staining(15). NCC-derived craniofacial bones and nasal cartilage andmandible (except for the interparietal bones) were dramaticallyablated or completely absent in the Wnt1Cre::SHP2�3–4/�3–4

embryos (Fig. 1C and D). The data demonstrate that NCC-autonomous SHP2 expression is necessary for normal craniofa-cial formation and underscore the importance of normal SHP2

activity in the migratory NCCs participating in craniofacialdevelopment.

Cardiac NCCs, a subpopulation of the cranial NCCs, originatefrom the lower hindbrain between the otic placode and fourthsomite. The cells contribute to the remodeling of arch arteries,septation of the OFT, closure of the ventricular septum, andinnervation of the cardiac ganglia (16–18). Because cardiovas-cular and craniofacial defects often occur concomitantly in NS,the cardiovascular phenotypes in the Wnt1Cre::SHP2�3–4/wt andWnt1Cre::SHP2�3–4/�3–4 embryos were analyzed (Fig. 2 andTable S2). In the Wnt1Cre::SHP2�3–4/wt embryos, craniofacialand cardiovascular phenotypes were completely normal, sug-gesting that a single allele of SHP2 is sufficient to allow NCCsto function in normal craniofacial and cardiovascular formation.In the Wnt1Cre::SHP2�3–4/�3–4 embryos, the gross anatomy ofthe atria and ventricles was normal (Fig. 2 A); strikingly, how-ever, all of the hearts exhibited persistent truncus arteriosus(PTA). Based on van Praagh’s PTA classification (19), 84.2% ofthe hearts had type II PTA (Fig. 2B) and 15.8% had type I PTA(Fig. 2C and D). Abnormal anatomy of the arch arteries wasobserved in 31.6% of the hearts. The Wnt1Cre::SHP2�3–4/�3–4

embryos demonstrated normal architecture of the atrial andventricular walls, but invariably exhibited ventricular septal defects(Fig. 2E and I). Atrioventricular valves were intact in theWnt1Cre::SHP2�3–4/�3–4 embryos (Fig. 2F and J). Further detailedanalyses of the PTA pathology in the Wnt1Cre::SHP2�3–4/�3–4

embryos were performed. Normally, the aortic and pulmonaryvalves consist of 3 leaflets (Fig. 2G and H); here, however, 90.0%of the Wnt1Cre::SHP2�3–4/�3–4 truncal valves contained 3 leaflets,and 10% (n � 10) had 4 leaflets (Fig. 2K and L). Longitudinal viewsshowed significant elongation and thickening of the truncal valvesof the Wnt1Cre::SHP2�3–4/�3–4 embryos compared with the aorticvalves in control littermates. The data indicate that NCC mutationscan result in a spectrum of defects associated with the diverse rolesof this progenitor population in multiple organ and system devel-opment, and underscore the importance of NCC SHP2 expressionin the normal development of both cardiovascular and craniofacialanatomy.

SHP2-Deficient NCCs Exhibit Altered Migration and Differentiation.SHP2 can regulate cell migration, proliferation, survival, anddifferentiation (20). NCCs can differentiate into a broad rangeof cell types once they reach their final destination. Here NCClineage was followed through breeding of our mice into either theROSA26 or EGFP indicator background. In normal mousedevelopment, NCCs begin to migrate after E8.5 and reach targetorgan primordia by E10. At E10, the SHP2-deficient NCCsappeared to be normally distributed in the craniofacial region,pharyngeal arches, and distal truncus compared with controls

Fig. 1. Craniofacial phenotypes of Wnt1Cre::SHP2�3–4/�3–4 embryos at E17.5. (A) Lateral view of the face from the control and Wnt1Cre::SHP2�3–4/�3–4 embryos.(B) Sagittal section of the head and neck (H&E staining). (C and D) Alcian blue and alizarin red staining of the skull. (C) Lateral view. (D) Upper view. t, tongue;nph, nasopharynx; fr, frontal bone of the skull; n, nasal bone; pm, premaxillary bone; m, mandible.

Nakamura et al. PNAS � July 7, 2009 � vol. 106 � no. 27 � 11271

MED

ICA

LSC

IEN

CES

Dow

nloa

ded

by g

uest

on

June

1, 2

020

(Fig. 3A), indicating that their initial proliferation and migrationpatterns were unaffected. A subpopulation of cardiac NCCscontinues to migrate and colonizes the proximal OFT andendocardial cushions, where aorticopulmonary septation occurs.At E11.5, NCCs of the Wnt1Cre::SHP2�3–4/�3–4 embryos stilldemonstrated normal migration to the proximal OFT (Fig. 3B);however, at E12.5, although the NCCs were still abundantlydistributed (Fig. 3C), sectioned material revealed significantlyreduced numbers of NCCs in the OFT cushions of the homozy-gous knockout hearts (Fig. 3D), culminating in the failure ofproper OFT septation. In the later embryonic stages, cardiacNCCs populate the tunica media of the aortic arch arteries and,together with endothelial cells and endoderm, contribute to theremodeling that results in the adult left-sided aortic arch vascularpattern (21). In E17.5 Wnt1Cre::SHP2�3–4/�3–4 hearts, NCCsappeared to be normally distributed in the carotid and rightbrachiocephalic artery and distal OFT, but were substantiallydecreased in the proximal OFT (Fig. 3E).

Cranial NCCs in Wnt1Cre::SHP2�3–4/�3–4 embryos appearedto migrate normally to the developing face at E10 and wereretained in the frontal bone and dura mater areas at E17.5 (Fig.3F and G). Because the frontal bone structures are defective inthe Wnt1Cre ::SHP2�3–4/�3–4 embryos (Fig. 1 D and E), weexplored whether cranial NCC differentiation into osteoblastswas altered. The osteoblast marker osteopontin was abundantlyexpressed in the control frontal bone, whereas no osteopontinexpression was observed in the Wnt1Cre::SHP2�3–4/�3–4 em-bryos (Fig. 3H), confirming the failure of normal differentiationof SHP2 null NCCs. Immunofluorescent staining was performedusing Ki-67 for cell proliferation and active caspase 3 for analysisof apoptosis. In sections from E9.5 pharyngeal arches and E12.5OFT, both Ki-67 and active caspase 3 staining were identical inWnt1Cre::SHP2�3–4/�3–4 embryos and controls, and this wasmaintained up to E12.5 (Fig. S2).

PTPN11 Ablation in NCCs Selectively Down-Regulates the ERK1/2Pathway. SHP2 can modulate the MAPK cascades (20), and weexamined SHP2’s several candidate downstream pathways in the

modified NCCs. ERK1/2 phosphorylation was severely affectedin the SHP2-deficient NCCs (Fig. 4A and B), but the activity ofother potentially affected signaling pathways, including theRhoA and JAK/STAT branches, remained unchanged (Fig. S3).Because inactivation of TGF-� signaling in NCCs can causedeficits similar to those noted earlier (22), we explored whetherSHP2 knockdown had an effect on TGF-� signaling. TGF-�activity, as assessed by phospho-Smad2/3 immunostaining, alsowas unchanged in the Wnt1Cre::SHP2�3–4/�3–4 embryos com-pared with controls (Fig. S3). These results indicate that theERK1/2 branch of the MAPK pathway is selectively affected inthe SHP2-deficient NCCs.

In normal mouse embryogenesis, ERK1/2 is transiently (until�E10.5) activated in some tissue primordium, including theneural crest, peripheral nervous system, blood vessels, and heart(23). The significance of ERK1/2 activation during early devel-opment remains obscure, however. Because ERK1/2 was selec-tively down-regulated in SHP2-deficient NCCs, and these cellsfailed to differentiate into osteoblasts, we tested the hypothesisthat ERK1/2 activation is necessary for NCC differentiationusing neural tube explant assays. Neural tubes from the cardiacneural crest region were isolated from E8.5 embryos and grownin culture. To inactivate ERK1/2, the MEK inhibitor U0126 wasadded for the first 48 h (Fig. 5A), after which the culture mediumwas changed and the NCCs were incubated for another 48 h.DMSO-treated control cardiac NCCs robustly differentiatedinto neurons and smooth muscle cells, as determined by immu-nofluorescent staining for TUJ1 and SMA, respectively (Fig.5B). In contrast, U0126-treated NCCs showed a dramatic re-duction in the number of TUJ1- and SMA-positive cells (Fig. 5Band C), underscoring the importance of ERK1/2 activation inthese differentiation processes.

We next determined whether ERK1/2 activation was sufficientto promote cell differentiation in the cultures. To reactivateERK1/2, adenovirus carrying constitutive active (ca)MEK1 wasused to infect the U0126-treated cultures for the first 48 h, afterwhich the cells were switched to normal culture medium andincubated for an additional 48 h. Increased levels of phospho-

A B C D

E F G H

I J K L

Fig. 2. Cardiovascular pathology in PTPN11 knockout mice. (A and B) Type II PTA in Wnt1Cre::SHP2�3–4/�3–4 hearts. Frontal views of a control (SHP2loxP/loxP; Left)versus a Wnt1Cre::SHP2�3–4/�3–4 heart at E17.5. (Magnification: �2.5.) Note that both atria were removed in (B). lca, left carotid artery; rca, right carotid artery;Aao, ascending aorta; PT/A, pulmonary trunk/artery. The arrow in (B) indicates the continued aorta/pulmonary artery present in the truncus arteriosus. (C andD) Type I PTA of Wnt1Cre::SHP2�3–4/�3–4 hearts at E17.5. (C) Frontal view, with both atria removed. (Magnification: �2.5.) (D) Transverse sections of the OFT ina Wnt1Cre::SHP2�3–4/�3–4 embryo. The cutting levels are shown in (C); the section, in (D). Ao, aorta; PT, pulmonary trunk. (E–L) H&E staining of E17.5 hearts. (Eand I) Longitudinal sections of the hearts. (Magnification: �10.) The arrowheads in (I) indicate a ventricular septal defect. (F and J) Longitudinal section of theatrioventricular valves. (Magnification: �10.) MV, mitral valve; TV, tricuspid valve. (G, H, K, and L) Transverse sections of the OFT valves. (Magnification: �20.)(G) and (H) show the aortic and pulmonary valves (AV and PV), respectively. Vl, valve leaflet

11272 � www.pnas.org�cgi�doi�10.1073�pnas.0902230106 Nakamura et al.

Dow

nloa

ded

by g

uest

on

June

1, 2

020

ERK1/2 (Fig. 5A) were accompanied by increases in SMA- andTUJ1-positive NCCs (Fig. 5B and C), suggesting that transientERK1/2 activation in NCCs is necessary for the differentiationof NCCs into diverse cell lineages during embryogenesis.

DiscussionERK1/2 is transiently activated during early embryogenesis (23),and ERK2 is critical for normal neural crest development (24).We and others have previously shown that mutations in SHP2associated with NS affect ERK1/2 signaling (25), and thus wethought it logical to explore the NCC-autonomous consequencesof SHP2 deficiency, particularly as it pertains to ERK1/2 acti-vation and the resultant effects on NCC migration, proliferation,and differentiation. Our data show that SHP2 deficiency inNCCs leads to decreased ERK1/2 activation, resulting in afailure of cell differentiation at diverse sites in the developingembryo with anatomical and functional deficits so severe as topreclude viability of the developing fetus.

We chose to study NCCs because of their migratory patternsand ability to give rise to many different cell types, reasoning thataltered SHP2 activity in a single precursor population, such asNCCs, might be responsible for some of the pathologies pre-senting at diverse sites in NS. Complete ablation of SHP2expression did result in decreased ERK1/2 activation in thepremigratory NCCs and, strikingly, influenced the developmentof both cardiac and cranial structures, 2 systems frequentlyaffected in NS (26).

The phenotype resembles other syndromic presentations. The22q11 deletion syndrome is the most common microdeletionsyndrome in humans, with an estimated incidence of 1 in 4,000live births. This syndrome comprises DiGeorge syndrome, ve-

locraniofacial syndrome, and conotruncal anomaly face syn-drome; �90% of the patients share hemizygous deletions ofproximal chromosome 22q11.2, most often encompassing a3-Mb region that includes �30 genes (27). The patients exhibitcraniofacial anomalies, thymus and parathyroid gland aplasia/hypoplasia, and cardiac OFT defects, thought to arise principallyfrom perturbed NCC development (28). In a mouse model,homozygous inactivation of Tbx1 results in a highly consistent22q11 deletion syndrome, and deletion of a single copy of Tbx1results in a partially penetrant 22q11 deletion syndrome pheno-type (29). Inactivation of the V-crk sarcoma virus CT10 onco-gene homolog (avian)-like gene, (Crkl), whose protein productactivates the RAS and JUN kinase-signaling pathways, alsoresults in a 22q11 deletion syndrome–like phenotype (30). TheWnt1Cre::SHP2�3–4/�3–4 mice in the present study exhibiteddefects consistent with those found in many patients sufferingfrom 22q11 deletion syndrome. Based on our data, we speculatethat some facets of the syndrome may arise from deficits in theNCCs.

Many of these diverse syndromes are linked by the commontheme of aberrant ERK signaling as a result of alterations inupstream effectors. For example, FGF8 encodes a polypeptidewith many important functions during mammalian development.By binding to its cognate receptor, FGF stimulates the ERK1/2cascade by promoting the association of the adaptor proteinSHP2 with small G proteins that are activators of Ras (31). Rasactivation can lead to increased ERK1/2 signaling. AlthoughFGF8 is not located on 22q11, FGF8 interacts with multipleproteins with genes located in or proximal to 22q11. FGFsignaling has been proposed to mediate TBX1’s role in pharyn-geal development (32). Similarly, CRKL and FGF8 interact such

Fig. 3. NCC fate-mapping analyses. Controls and homozygous nulls were crossed into the ROSA26 background to allow tracing of NCC lineages by�-galactosidase staining. (A) �-galactosidase–stained embryos at E10. (Magnification: �4.) Arrowheads indicate the cells in the conotruncal regions. (B) Anteriorview of E11.5 hearts. (Magnification: �5.) AA, aortic arch; pOFT, proximal outflow tract. (C) Anterior view of E12.5 hearts. (Magnification: �5.) (D) Longitudinalsection of the OFT at E12.5. (Magnification: �20.) Ao, aorta; PT, pulmonary trunk. (E) OFT and arch arteries. (Magnification: �6.) Rca, right carotid artery; lca,left carotid artery; Ao, aorta; PT, pulmonary trunk. (F) �-galactosidase staining of an E17.5 skull showing the contribution of NCC-derived cell lineages. FB, frontalbone; PB, parietal bone. (G) Sagittal section of the frontal and parietal bones. The numbers refer to the section sites shown in (A). DM, dura mater, FB, frontalbone, PB; parietal bone. (H) Osteopontin (OPN) immunostaining of the frontal bone.

Nakamura et al. PNAS � July 7, 2009 � vol. 106 � no. 27 � 11273

MED

ICA

LSC

IEN

CES

Dow

nloa

ded

by g

uest

on

June

1, 2

020

that CRKL can regulate the expression of certain FGF8 targetgenes in the pharyngeal arches. Intriguingly, CRKL deficiencydisrupts ERK1/2 activation (33). As is the case for theWnt1Cre::SHP2�3–4/�3–4 embryos, mice with reduced or elimi-nated FGF8 expression exhibit reduced ERK1/2 phosphoryla-tion. These animals have craniofacial and cardiovascular defectssimilar to those noted in TBX1- and CRKL-mutant mice,respectively (34, 35).

Recent genetic data directly implicate altered ERK activitywith the clinical presentation of these syndromes. ERK2 ispositioned distal to and outside the 3-Mb region of 22q11, andrecent data obtained from patients with a small (�1 Mb) distal22q11.2 microdeletion showed an association between haploin-sufficiency for MAPK1/ERK2 and neural crest deficits. Thesepatients exhibited some of the classic defects of the 22q11deletion syndrome spectrum, including craniofacial andconotruncal cardiac anomalies (24), and the authors went on toshow that NCC-specific deletion of ERK2 or NCC-specific

inactivation of upstream components of the pathway, MEK1/MEK2 and B-Raf/C-Raf, also led to similar cardiac and cranio-facial defects. In the present study, the SHP2-null selectivelyinactivated the ERK1/2 pathway, and the phenotypes of theWnt1Cre::SHP2�3–4/�3–4 embryos were mostly consistent withthose of both RAF/MEK/ERK-inactivated mice and TBX1-,CRKL-, and FGF8-mutant mice. These findings suggest thatmany aspects of the pathogenesis of the 22q11 deletion syndromemay be related to perturbation of a common genetic pathwaynecessary for the activation or correct level of ERK1/2 signaling.Even with the limited data currently available, it is apparent thatthe RAS/MAPK syndromes can arise as a result of multiplemutations in different genes that encode both proximal anddistal players, and that multiple syndromes (e.g., Noonan,Costello) can arise through different mutations in the same ordifferent genes (3).

LS is an autosomal dominant disorder; the acronymic name‘‘LEOPARD’’ refers to its major features of lentigines, electro-cardiographic conduction abnormalities, ocular hypertelorism,pulmonic stenosis, abnormal genitalia, retardation of growth,and sensorineural deafness. SHP2 mutations in LS are clusteredin the PTP domain and lead to decreased SHP2 activity. LSalleles exhibit substantial ERK1/2 activation in vitro (5, 36),consistent with our observations in the SHP2-ablated NCCs. Wefirst hypothesized that a SHP2 null in NCCs might mimic someaspects of LS; unexpectedly, however, the mice more closelyphenocopied the 22q11 deletion syndrome. A potential expla-nation for this discrepancy is that the complete absence of SHP2may have different consequences than the expression of a

Fig. 4. ERK1/2 phosphorylation is decreased in Wnt1Cre::SHP2�3–4/�3–4 NCCs.(A) phospho-ERK1/2 immunostaining of the E9.5 pharyngeal arch. The bottompanel corresponds to a magnified portion of the bounded areas in the panelsimmediately above; arrowheads identify NCCs containing activated phos-phorylated ERK1/2. (B) Quantification of phospho-ERK1/2–positive NCCs. Atleast 5,000 NCCs were analyzed from 5 embryos. *P � .0001.

Fig. 5. Transient ERK1/2 activation of cardiac NCCs restores their ability toexpress neural and smooth muscle markers. (A) Activated ERK1/2 levels inneural tube cultures. (B) SMA (Top) and TUJ1 (Bottom) immunostaining ofcardiac NCCs. (Magnification: �20.) ca, constitutively active. (C) Quantificationof SMA- and TUJ1-positive NCCs. At least 3,000 NCCs were analyzed in eachgroup. *P � .0001.

11274 � www.pnas.org�cgi�doi�10.1073�pnas.0902230106 Nakamura et al.

Dow

nloa

ded

by g

uest

on

June

1, 2

020

LS-causing dominant-negative mutant; for example, the mutantprotein expressed may still interact with a set or subset ofadaptor/scaffolding proteins, altering critical protein–proteininteractions. A possible alternative explanation is that differ-ences in the magnitude of ERK1/2 reduction might lead todistinct craniofacial and cardiovascular phenotypes. Acting syn-ergistically, the ERK1 and ERK2 knockouts in NCCs exacerbatecraniofacial defects (24). Resolution of these discrepancies willrequire creating a knockin LS mutant mouse and analyzing theresulting development of the craniofacial and cardiovascularsystems.

Animal models developed to study the RAS/MAPK syn-dromes include mouse models with systemic knockouts of SHP2(37), as well as knockin models carrying SHP2 mutations foundin humans (38). Recently, both activating and inactivating SHP2mutations have been studied in zebrafish (39). Morpholino-mediated SHP2 knockdown resulted in early defects in gastru-lation, precluding detailed studies of organogenesis; nonetheless,it was apparent that loss of function led to defective cellularmovement. Similarly, expression of either LS or NS mutants alsoresulted in defects in cell convergence and extension, and theembryos also displayed both craniofacial and cardiovasculardefects (39). Our data confirm that SHP2 levels are functionallyimportant in the NCCs, modulating ERK1/2 activity and affect-ing development of the OFT and frontal skull structures. NCC-autonomous SHP2 activity is critical for normal development in

both the heart and skull, and there appears to be a relativelynarrow window of activity that is conducive to normal develop-ment, with too little or too much SHP2 activity leading toaberrant development. These data are consistent with the hy-pothesis that alterations in the NCCs as a result of altered SHP2activity play a major role in the pathogenic presentation ofvarious syndromic diseases. A detailed exploration of the exactalterations in migration, proliferation, and differentiation un-derlying the pathogenesis in the various organ systems would bevaluable.

Materials and MethodsGeneration of Mice. Animal use was in accordance with institutional guide-lines, and all experiments were reviewed and approved by the institutionalAnimal Care Committee. All lines of transgenic and gene-targeted mice weremade in or backcrossed onto a C57BL/6 background for at least 15 generationsbefore being used in the crosses analyzed. The following sequences were usedfor genotyping: Wnt1Cre, sense 5�-ATTCTCCCACCGTCAGTACG-3�, antisense5�-CGTTTTCTGAGCATACCTGGA-3�; SHP2 knockout, sense 5�-CTGGAAGAG-CAGTCAGTCA GTGCT-3�, antisense 5�-CCACTCACCTTGTCATGTAGTAACTC-3�;ROSA26R, sense 5�-TGGGGAATGAATCAGGCCACGG-3�, antisense 5�-GCGT-GGGCGTATTCGCCAAGGA-3�; EGFP, sense 5�-ACGTAAACGGCCACAAGT TCA-3�, antisense 5�-GCTGTT GTAGTTGTACTCCAGGT-3�. Additional methods aredetailed in the SI Materials and Methods.

ACKNOWLEDGMENTS. This work was supported by National Institutes ofHealth Grants P01HL69799, P50HL07701, P01HL059408, and R01HL087862(to J.R.).

1. Rosamond W, et al. (2008) Heart disease and stroke statistics, 2008 update: A reportfrom the American Heart Association Statistics Committee and Stroke Statistics Sub-committee. Circulation 117:e25–146.

2. Schubbert S, Bollag G, Shannon K (2007) Deregulated Ras signaling in developmentaldisorders: New tricks for an old dog. Curr Opin Genet Dev 17:15–22.

3. Aoki Y, et al. (2008) The RAS/MAPK syndromes: Novel roles of the RAS pathway inhuman genetic disorders. Hum Mutat 29:992–1006.

4. Bentires-Alj M, Kontaridis MI, Neel BG (2006) Stops along the RAS pathway in humangenetic disease. Nat Med 12:283–285.

5. Kontaridis MI, et al. (2006) PTPN11 (Shp2) mutations in LEOPARD syndrome havedominant negative, not activating, effects. J Biol Chem 281:6785–6792.

6. Tartaglia M, et al. (2007) Gain-of-function SOS1 mutations cause a distinctive form ofNoonan syndrome. Nat Genet 39:75–79.

7. Kontaridis MI, et al. (2008) Deletion of Ptpn11 (Shp2) in cardiomyocytes causes dilatedcardiomyopathy via effects on the extracellular signal–regulated kinase/mitogen-activated protein kinase– and RhoA-signaling pathways. Circulation 117:1423–1435.

8. Tang TL, et al. (1995) The SH2-containing protein-tyrosine phosphatase SH-PTP2 isrequired upstream of MAP kinase for early Xenopus development. Cell 80:473–483.

9. Yang W, et al. (2006) An Shp2/SFK/Ras/Erk signaling pathway controls trophoblast stemcell survival. Dev Cell 10:317–327.

10. Delfino-Machin M, Chipperfield TR, Rodrigues FS, Kelsh RN (2007) The proliferatingfield of neural crest stem cells. Dev Dyn 236:3242–3254.

11. Trainor PA (2005) Specification of neural crest cell formation and migration in mouseembryos. Semin Cell Dev Biol 16:683–693.

12. Hof P, et al. (1998) Crystal structure of the tyrosine phosphatase SHP-2. Cell 92:441–450.13. Nakamura T, Colbert MC, Robbins J (2006) Neural crest cells retain multipotential

characteristics in the developing valves and label the cardiac conduction system. CircRes 98:1547–1554.

14. Chai Y, Maxson RE, Jr. (2006) Recent advances in craniofacial morphogenesis. Dev Dyn235:2353–2375.

15. Ito Y, et al. (2003) Conditional inactivation of Tgfbr2 in cranial neural crest causes cleftpalate and calvaria defects. Development 130:5269–5280.

16. Hutson MR, Kirby ML (2003) Neural crest and cardiovascular development: A 20-yearperspective. Birth Defects Res C Embryo Today 69:2–13.

17. Kirby ML, Stewart DE (1983) Neural crest origin of cardiac ganglion cells in the chickembryo: Identification and extirpation. Dev Biol 97:433–443.

18. Snider P, Olaopa M, Firulli AB, Conway SJ (2007) Cardiovascular development and thecolonizing cardiac neural crest lineage. ScientificWorldJournal 7:1090–1113.

19. Van Praagh R, Van Praagh S (1965) The anatomy of common aorticopulmonary trunk(truncus arteriosus communis) and its embryologic implications: A study of 57 necropsycases. Am J Cardiol 16:406–425.

20. Neel BG, Gu H, Pao L (2003) The ‘‘Shp’’ing news: SH2 domain–containing tyrosinephosphatases in cell signaling. Trends Biochem Sci 28:284–293.

21. LeDourin NM, Kalcheim G (1999) The Neural Crest (Cambridge Univ. Press, Cambridge),2nd ed.

22. Wurdak H, et al. (2005) Inactivation of TGFbeta signaling in neural crest stem cells leadsto multiple defects reminiscent of DiGeorge syndrome. Genes Dev 19:530–535.

23. Corson LB, Yamanaka Y, Lai KM, Rossant J (2003) Spatial and temporal patterns of ERKsignaling during mouse embryogenesis. Development 130:4527–4537.

24. Newbern J, et al. (2008) Mouse and human phenotypes indicate a critical conservedrole for ERK2 signaling in neural crest development. Proc Natl Acad Sci USA 105:17115–17120.

25. Nakamura T, et al. (2007) Mediating ERK 1/2 signaling rescues congenital heart defectsin a mouse model of Noonan syndrome. J Clin Invest 117:2123–2132.

26. Allanson JE (2007) Noonan syndrome. Am J Med Genet C Semin Med Genet 145C:274–279.

27. Shprintzen RJ (2008) Velo-cardio-facial syndrome: 30 years of study. Dev Disabil Res Rev14:3–10.

28. Schinke M, Izumo S (1999) Getting to the heart of DiGeorge syndrome. Nat Med5:1120–1121.

29. Jerome LA, Papaioannou VE (2001) DiGeorge syndrome phenotype in mice mutant forthe T-box gene, Tbx1. Nat Genet 27:286–291.

30. Guris DL, et al. (2001) Mice lacking the homologue of the human 22q11.2 gene CRKLphenocopy neurocristopathies of DiGeorge syndrome. Nat Genet 27:293–298.

31. Bottcher RT, Niehrs C (2005) Fibroblast growth factor signaling during early vertebratedevelopment. Endocr Rev 26:63–77.

32. Vitelli F, et al. (2002) A genetic link between Tbx1 and fibroblast growth factorsignaling. Development 129:4605–4611.

33. Moon AM, et al. (2006) Crkl deficiency disrupts FGF8 signaling in a mouse model of22q11 deletion syndromes. Dev Cell 10:71–80.

34. Abu-Issa R, et al. (2002) FGF8 is required for pharyngeal arch and cardiovasculardevelopment in the mouse. Development 129:4613–4625.

35. Park EJ, et al. (2006) Required, tissue-specific roles for FGF8 in outflow tract formationand remodeling. Development 133:2419–2433.

36. Tartaglia M, et al. (2006) Diversity and functional consequences of germline andsomatic PTPN11 mutations in human disease. Am J Hum Genet 78:279–290.

37. Chen B, et al. (2000) Mice mutant for Egfr and Shp2 have defective cardiac semilunarvalvulogenesis. Nat Genet 24:296–299.

38. Araki T, et al. (2004) Mouse model of Noonan syndrome reveals cell type– and genedosage–dependent effects of Ptpn11 mutation. Nat Med 10:849–857.

39. Jopling C, van Geemen D, den Hertog J (2007) Shp2 knockdown and Noonan/LEOPARDmutant Shp2-induced gastrulation defects. PLoS Genet 3:e225.

Nakamura et al. PNAS � July 7, 2009 � vol. 106 � no. 27 � 11275

MED

ICA

LSC

IEN

CES

Dow

nloa

ded

by g

uest

on

June

1, 2

020