Neural Crest Migration and Mouse Models of Congenital Heart Disease A.D. GITLER, C.B. BROWN, L. KOCHILAS, J. LI,

AND

J.A. EPSTEIN

Department of Medicine, University of Pennsylvania Health System, Philadelphia, Pennsylvania 19104

Congenital heart disease is common, affecting nearly 1% of live births. Perhaps one-third of the cases of congenital heart disease involve abnormal morphogenesis of the outflow tract of the heart and/or abnormal patterning of the great vessels (Driscoll 1994; Goldmuntz et al. 1998). The development of these structures is influenced by migrating neural crest cells that arise from the dorsal neural tube and populate the pharyngeal arches and the heart (Kirby et al. 1983). Ablation studies in chick embryos suggest that neural crest migration defects can result in congenital heart disease. Recently, however, analyses of a series of mouse models with genetic forms of congenital heart disease that resemble human disease suggest that migration defects are uncommon. Newly developed molecular markers and fate-mapping techniques suggest that functional defects of post-migratory neural crest cause some forms of common structural cardiovascular disorders. These defects can be cell autonomous or can arise from defects in cells surrounding the neural crest migration pathways. CARDIAC NEURAL CREST During late stages of mammalian and avian cardiogenesis, the looped heart tube undergoes a series of septation events that result in the establishment of parallel pulmonary and systemic circulations (Fishman and Chien 1997). At the same time, the aortic arch arteries, which arise as paired sets of bilaterally symmetric vessels, undergo dramatic remodeling in order to produce the asymmetric adult vascular system (Epstein and Buck 2000). Landmark studies performed in the 1980s identified a critical requirement for neural crest cells in these processes (Kirby et al. 1983; Kirby 1988). Neural crest cells arise from the dorsal neural tube and migrate throughout the developing embryo. They function as pluripotent progenitor cells and can differentiate into most mesodermal cell types (Hall 1999; Le Douarin and Kalcheim 1999). A subset of neural crest cells migrates from the level of the first three somites and contributes significant cell mass to the developing branchial arches. DiI labeling studies and quail–chick chimera analyses indicate that some of these migrating cells populate the outflow tract of the heart. Most significantly, ablation of these cells in chick embryos before they emerge from the neural tube results in predictable forms of con-

genital heart disease, including persistent truncus arteriosus, in which the aorta and pulmonary arteries fail to arise as distinct vessels due to the absence of outflow tract septation (Kirby et al. 1983). Other defects include doubleoutlet right ventricle and interruption of the aortic arch. These important experiments suggest that genetic defects which result in the absence of neural crest migration in mammals may cause congenital heart disease. In mammals, however, studies to track neural crest migration or to perform ablation studies have been more difficult than in avian species due to technical considerations and to the relative dearth of reliable molecular markers. However, a number of mouse models of congenital heart disease involving the outflow tract of the heart have arisen either spontaneously or via targeted mutation of specific genes (Epstein 1996, 2001; Epstein and Buck 2000). Our laboratory and others have developed new markers that are specific for migrating neural crest cells and that permit the accurate identification of these cells in normal embryos and in mutant strains (Waldo et al. 1999; Yamauchi et al. 1999; Jiang et al. 2000; Li et al. 2000; Brown et al. 2001). The surprising finding that is emerging from the analysis of a series of murine models with outflow tract and aortic arch defects is that neural crest migration is generally preserved. None of these models is caused by a complete loss of neural crest migration that would be analogous to the chick ablation models. Pax3 FUNCTION AND CARDIAC DEFECTS IN Splotch mice Homozygous mutations in the gene encoding the transcription factor Pax3 result in congenital heart disease which closely resembles that seen in chick embryos after neural crest ablation (Franz 1989; Epstein 1996). Pax3–/– embryos succumb at embryonic day 13.5 (E13.5) with persistent truncus arteriosus and also show defects in other neural-crest-derived structures, including peripheral ganglia. Pax3 is expressed in the dorsal neural tube beginning as early as E8.5, before cardiac neural crest cells have delaminated and initiated migration toward the branchial arches (Goulding et al. 1991). Expression is extinguished shortly after migration has initiated (Epstein et al. 2000). Hence, it was attractive to assume that Pax3 was required for cardiac neural crest migration (Moase

Cold Spring Harbor Symposia on Quantitative Biology, Volume LXVII. © 2002 Cold Spring Harbor Laboratory Press 0-87969-678-8/02.

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and Trasler 1991, 1992) and that lack of migration resulted in cardiovascular defects. Some early studies indicated abnormal migratory behavior of cardiac neural crest cells derived from Splotch embryos (Moase and Trasler 1990), and later studies appeared to confirm these results (Conway et al. 1997). However, subsequent reanalysis of these latter studies suggests that the migratory population dependent on Pax3 is actually a pool of hypoglossal and hypaxial muscle progenitors rather than cardiac neural crest (Epstein et al. 2000). We reexamined neural crest migratory behavior in Splotch embryos using a transgenic mouse line to aid in the identification of neural crest cells. A portion of the connexin 43 (Cx43) gene upstream regulatory region was used to drive expression of lacZ and was found to direct expression to neural crest cells in developing embryos (Waldo et al. 1999). We crossed Cx43-lacZ mice with Splotch mice and examined neural crest patterning at E12.5 in wild-type and homozygous Splotch embryos. Interestingly, we found significant numbers of labeled cells that had migrated throughout the branchial arches, encased the aortic arch arteries, and invaded the outflow tract of the heart in both wild-type and mutant embryos (Epstein et al. 2000). The precise patterning of neural crest derivatives in the heart was not identical since the outflow tract septum was poorly formed or absent in mutant embryos, and there appeared qualitatively to be fewer labeled cells at the most distal zones of migration in Splotch embryos. In addition, in some embryos, labeled cells were not as tightly clustered in the region of the endocardial cushion in mutant embryos when compared to wild type. Nevertheless, the unequivocal findings from these studies were that neural crest migration is not entirely dependent on Pax3 function, and the Splotch cardiac phenotype including persistent truncus arteriosus is not due to absence of neural crest cells in the heart. Rather, the defect is more subtle, stochastic, or related to postmigratory neural crest function. Cre-lox APPROACHES TO FATE-MAP NEURAL CREST We have taken an alternative approach to follow the fate of Pax3-expressing neural crest cells as they migrate throughout the embryos and undergo differentiation. We have identified a portion of the Pax3 upstream genomic region that is sufficient to recapitulate Pax3 expression in the dorsal neural tube while omitting expression in other domains of endogenous Pax3 expression such as the somite (Li et al. 1999). Like Pax3 itself, expression of a lacZ transgene driven by this regulatory region declines shortly after migration of neural crest cells initiates. However, using Cre-lox approaches, we have been able to indelibly label neural crest derivatives that expressed the Pax3 transgene earlier during development (Li et al. 2000). We characterized Pax3-Cre mice and demonstrated expression of Cre recombinase in the dorsal neural tube, although the lines of mice characterized to date exhibit ectopic expression in the caudal regions in mesenchymal tissue adjacent to the neural tube. By crossing

Pax3-Cre mice with Cre reporter mice, such as R26R mice in which lacZ expression is initiated by Cre-mediated recombination, cells that express Pax3-Cre are labeled by lacZ expression. Since lacZ is driven by a ubiquitous promoter in R26R mice, once lacZ expression is activated, it will continue to be expressed for the lifetime of that cell and by all daughter cells derived from that cell. Hence, Pax3-expressing neural crest precursors are fate-mapped, and their derivatives can be identified. We have used this system to fate-map Pax3-expressing neural crest precursors in Splotch embryos. These studies confirm the observation that neural crest migrates through the branchial arches and populates the heart in both wild-type and mutant embryos (Fig. 1). Moreover, Pax3-expressing precursors give rise to smooth muscle cells in the aortic arches and ductus arteriosus, and they compose the vast majority of the aorto-pulmonary septum in the outflow tract (Epstein and Buck 2000). Sema3C DEFICIENCY AND NEURAL-CRESTRELATED CARDIAC DEFECTS Semaphorins represent a family of related cell-surface and secreted molecules that play important roles in mediating repulsive guidance cues during central nervous system axonal migration (Yu and Kolodkin 1999; Tamagnone and Comoglio 2000). Certain secreted semaphorin molecules mediate growth cone collapse and hence direct patterning of neuronal circuitry. Inactivation of Semaphorin 3C (Sema3C) in the mouse unexpectedly resulted in aortic arch and cardiac outflow tract defects that included interruption of the aortic arch and persistent truncus arteriosus (Feiner et al. 2001). Sema3C is a secreted semaphorin and is presumed to bind to a heterodimeric receptor composed of a plexin subunit and a neuropilin subunit. Sema3C is expressed in the non-neural-crest-derived mesenchyme of the branchial arches and is likely to have a non-cell-autonomous function that results in neural crest abnormalities and cardiovascular defects (Feiner et al. 2001). We sought to identify a potential Sema3C receptor expressed by migrating neural crest cells. Although a number of molecules have been suggested as markers of migrating cardiac neural crest in mammals, none has been definitively proven to label these cells. Hence, we were aided considerably by the availability of the Cre-lox fatemapping system described above in order to identify postmigratory neural crest cells while costaining for potential semaphorin receptors. This approach allowed the identification of PlexinA2 expression by migrating cardiac neural crest (Brown et al. 2001). PlexinA2 is expressed by two streams of cells invading the cardiac outflow tract and forming the aorto-pulmonary septum. This pattern appears identical to that of fate-mapped Pax3-expressing neural crest cells in the heart. Because semaphorins are closely linked with regulation of migration, we examined the patterning of cardiac neural crest cells in wild-type and Sema3C-deficient embryos. Despite the absence of Sema3C, cardiac neural crest was able to find the proper migratory routes to the

CARDIAC NEURAL CREST MIGRATION

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Figure 1. Cardiac neural crest cells populate the cardiac outflow tract and great vessels. (A) Fate mapping was performed using mice that express Cre recombinase in Pax3-expressing neural crest precursors and R26R reporter mice. Neural crest derivatives are labeled blue. E11.5 heart shows neural crest cells in the cardiac outflow tract (arrow). (B) At E14.5, neural crest cells can be seen encasing the great vessels including the right common carotid (RCC), left common carotid (LCC), and aortic arch (AA) arteries. Neural crest cells in the cardiac outflow tract (black arrow) are partially obscured by myocardium. (LV) Left ventricle. (RV) Right ventricle.

cardiac outflow tract, and the overall patterning of cardiac neural crest was unchanged. We did note subtle deficiencies in the maximal distance that cardiac neural crest migrated into the outflow tract, suggesting that subtle migratory defects may occur (Brown et al. 2001; Feiner et al. 2001). Migration in the region of the aortic arch arteries within the branchial arches was grossly unaffected despite the fact that aortic arch interruption developed in these embryos. These results suggest that defects of neural crest derivatives, other than migration defects, may account for some or all of the phenotypes seen in Sema3C-deficient mice. Perhaps semaphorin signaling affects differentiation or survival of this cell population. TYPE I NEUROFIBROMATOSIS GENE AND DOUBLE-OUTLET RIGHT VENTRICLE The type I neurofibromatosis gene (NF1) encodes a large intracellular protein capable of down-regulating Ras signaling (Cichowski and Jacks 2001). Mutation of this tumor suppressor gene in humans leads to type I neurofibromatosis, which is characterized by benign and malignant tumors of neural crest origin. Patients are heterozygotes and sustain a “second hit” in affected somatic tissues. In mice, heterozygous mutation of NfI also leads to a predisposition to cancer (Brannan et al. 1994; Jacks et al., 1994). Homozygous mutation leads to embryonic lethality, and affected embryos display congenital heart disease. Heart defects include double-outlet right ventricle, in which both the pulmonary artery and the aorta arise from the right ventricle (Brannan et al. 1994; Jacks et al.

1994; Lakkis and Epstein 1998). This abnormality is associated with a ventricular septal defect allowing blood to exit the left ventricle. Double-outlet right ventricle is also seen in chick embryos after ablation of premigratory neural crest cells. The association of type I neurofibromatosis with neural crest tumors, and the association of double-outlet right ventricle with neural crest ablation, suggested that congenital heart disease in Nf1–/– embryos was due to a defect in cardiac neural crest cells (Brannan et al. 1994; Jacks et al. 1994). Therefore, we examined neural crest migration in wild type and Nf1–/– embryos using molecular markers and Cre-lox fate-mapping systems. We also examined affected embryos for other signs of neuralcrest-related cardiovascular defects. In 1 of 20 Nf1–/– E13.5 embryos examined, we identified abnormal remodeling of the aortic arch arteries resulting in retro-esophageal right sublclavian artery, a defect associated with inappropriate regression of the right fourth aortic arch segment (Fig. 2). Interestingly, we identified a low incidence of the identical defect in Nf1 embryos in which the Nf1 gene was deleted only in neural crest cells using a Cre-lox approach. However, we did not detect any instances of more severe aortic arch or neural crest defects such as persistent truncus arteriosus or interruption of the aortic arch. Nevertheless, all mutant embryos exhibited double-outlet right ventricle. Our preliminary results also suggest that neural crest migration in Nf1 mutant embryos is intact. Using Cre-lox approaches described above, we followed neural crest cells throughout their migration along the aortic arch seg-

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Figure 2. Retroesophageal right subclavian artery (black arrows) is present in a minority of Nf1–/– E13.5 embryos (A) and in an embryo lacking Nf1 only in neural crest cells produced by a Cre-lox approach (B). Transverse sections stained with H&E at the level of the cardiac outflow tract are shown. Ventral is at the top; dorsal at the bottom. The right subclavian artery normally travels dorsal to the esophagus (Es) and trachea (Tr). These sections are at the level of the pulmonary valve and pulmonary artery (PA) and the ductus arteriosus is patent, allowing continuity between the PA and the right subclavian which is originating ectopically from the descending aorta near the junction of the ductus arteriosus and the aorta.

ments and into the cardiac outflow tract. In both wild type and Nf1 mutants, abundant neural crest cells were identified in the appropriate locations. Of note, double-outlet right ventricle is completely penetrant in Nf1 mutant embryos, suggesting that, although this defect can be caused by an absence of neural crest migration, it can also occur through other mechanisms. MOUSE MODELS OF DiGEORGE SYNDROME AND Tbx1 DiGeorge syndrome is a common human genetic disorder occurring in 1 of 4000 live births (Driscoll 1994; Epstein and Buck 2000; Epstein 2001). The syndrome is characterized by cardiac outflow tract defects, parathyroid deficiency, thymus and thyroid abnormalities, and craniofacial defects. Learning difficulties can also be associated. Many of the affected organs have neural crest contributions, suggesting that DiGeorge syndrome may be related to abnormal neural crest development. In many cases, deletions on human chromosome 22q11 have been associated with familial and sporadic cases (Goldmuntz et al. 1998). Recently, a series of mouse models of DiGeorge syndrome have been created that take advantage of the fact that the commonly deleted region of human chromosome 22 is homologous to a conserved region of mouse chromosome 11 (Lindsay et al. 1999, 2001; Epstein 2001;

Merscher et al. 2001; Schinke and Izumo 2001). Heterozygous deletion of a ~1.5-Mb region, including about 20 genes, results in mice with cardiac outflow tract defects, including interruption of the aortic arch, retroesophageal subclavian artery, and related defects (Lindsay et al. 1999, 2001; Merscher et al. 2001). These mice also have parathyroid and thymus defects reminiscent of human patients. Hence, these mice provide a reasonable animal model for at least some aspects of DiGeorge syndrome. A series of elegant complementation experiments using additional mouse lines that contained extra copies of some of the deleted genes allowed researchers to focus on a small group of genes within the deleted region that were responsible for the cardiovascular abnormalities (Lindsay et al. 2001; Merscher et al. 2001). Subsequent analysis identified Tbx1 as the critical gene, and heterozygous targeted deletion of Tbx1 results in similar cardiovascular abnormalities (Jerome and Papaioannou 2001; Lindsay et al. 2001; Merscher et al. 2001). Interestingly, Tbx1 is apparently not expressed by cardiac neural crest cells, but rather is expressed by the core mesenchyme of the pharyngeal arches through which cardiac neural crest migrates. We asked whether Tbx1 haploinsufficiency affected neural crest migration. We examined the expression of PlexinA2, and we used Cre-lox strategies to examine neural crest patterning in wild-type and Tbx1+/– litter-

CARDIAC NEURAL CREST MIGRATION mates. We were unable to detect any differences in neural crest migration or location or gene expression in affected embryos, despite the evidence for aortic arch artery abnormalities. Hence, we conclude that wild-type expression of Tbx1 is not required for normal cardiac neural crest migration, and the cardiovascular defects seen in these mouse models of DiGeorge syndrome are not related to absence of neural crest cells in the pharyngeal region or the heart. Additional data from our lab and others, however, suggest that neural crest differentiation is abnormal in mouse models of DiGeorge syndrome. Cardiac neural crest cells encasing the aortic arch arteries invest the medial layer of these vessels and differentiate into smooth muscle. This differentiation process is deficient in DiGeorge models, and early markers of smooth muscle are reduced or absent in specific aortic arch segments including the 4th aortic arch artery. Since Tbx1 is expressed by adjacent mesenchyme, these results suggest a model in which Tbx1 induces the secretion of a critical growth factor that is required for proper survival or differentiation of nearby neural crest derivatives. CONCLUSIONS In summary, recent advances in genetics and cell biology have allowed the identification of new markers for cardiac neural crest cells and for genetic mechanisms for performing fate-mapping studies in the mouse. These tools have permitted the further evaluation of a series of mouse models of congenital heart disease that are associated with neural crest defects. Despite the fact that earlier studies in chick embryos suggested that neural crest migration defects would be associated with congenital heart disease, the surprising finding that is emerging from ongoing studies is that neural crest migration is relatively preserved in several diverse models of outflow tract and aortic arch disorders. It now appears likely that gross migration defects are relatively uncommon, whereas functional defects of postmigratory neural crest cells may be more common. These defects may involve survival, proliferation, and/or differentiation, and are likely to depend on critical interactions with non-neural-crest-derived neighboring cells. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health to J.A.E., C.B.B., and L.K. and from the W.W. Smith Foundation (J.A.E.) and the American Heart Association (J.A.E.). We are grateful to our collaborators Jonathan Raper, Luis Parada, and Cecilia Lo. REFERENCES Brannan C.I., Perkins A.S., Vogel K.S., Ratner N., Nordlund M.L., Reid S.W., Buchberg A.M., Jenkins N.A., Parada L.F., and Copeland N.G. 1994. Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues. Genes Dev. 8: 1019.

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Brown C.B., Feiner L., Lu M.M., Li J., Ma X., Webber A.L., Jia L., Raper J.A., and Epstein J.A. 2001. PlexinA2 and semaphorin signaling during cardiac neural crest development. Development 128: 3071. Cichowski K. and Jacks T. 2001. NF1 tumor suppressor gene function: Narrowing the GAP. Cell 104: 593. Conway S., Henderson D., and Copp A. 1997. Pax3 is required for cardiac neural crest migration in the mouse: Evidence from the splotch (Sp2H) mutant. Development 124: 505. Driscoll D.A. 1994. Genetic basis of DiGeorge and velocardiofacial syndromes. Curr. Opin. Pediatr. 6: 702. Epstein J.A. 1996. Pax3, neural crest and cardiovascular development. Trends Cardiovasc. Med. 6: 255. _______ . 2001. Developing models of DiGeorge syndrome. Trends Genet. 17: S13. Epstein J.A. and Buck C.A. 2000. Transcriptional regulation of cardiac development: Implications for congenital heart disease and DiGeorge syndrome. Pediatr. Res. 48: 717. Epstein J.A., Li J., Lang D., Chen F., Brown C.B., Jin F., Lu M.M., Thomas M., Liu E., Wessels A., and Lo C.W. 2000. Migration of cardiac neural crest cells in Splotch embryos. Development 127: 1869. Feiner L., Webber A.L., Brown C.B., Lu M.M., Jia L., Feinstein P., Mombaerts P., Epstein J.A., and Raper J.A. 2001. Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption. Development 128: 3061. Fishman M.C. and Chien K.R. 1997. Fashioning the vertebrate heart: Earliest embryonic decisions. Development 124: 2099. Franz T. 1989. Persistent truncus arteriosus in the Splotch mutant mouse. Anat. Embryol. 180: 457. Goldmuntz E., Clark B.J., Mitchell L.E., Jawad A.F., Cuneo B.F., Reed L., McDonald-McGinn D., Chien P., Feuer J., Zackai E.H., Emanuel B.S., and Driscoll D.A. 1998. Frequency of 22q11 deletions in patients with conotruncal defects. J. Am. Coll. Cardiol. 32: 492. Goulding M.D., Chalepakis G., Deutsch U., Erselius J.R., and Gruss P. 1991. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J. 10: 1135. Hall B.K. 1999. The neural crest in development and evolution. Springer, New York. Jacks T., Shih T.S., Schmitt E.M., Bronson R.T., Bernards A., and Weinberg R.A. 1994. Tumour predisposition in mice heterozygous for a targeted mutation in Nf1. Nat. Genet. 7: 353. Jerome L.A. and Papaioannou V.E. 2001. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat. Genet. 27: 286. Jiang X., Rowitch D.H., Soriano P., McMahon A.P., and Sucov H.M. 2000. Fate of the mammalian cardiac neural crest. Development 127: 1607. Kirby M.L. 1988. Nodose placode contributes autonomic neurons to the heart in the absence of cardiac neural crest. J. Neurosci. 8: 1089. Kirby M.L., Gale T.F., and Stewart D.E. 1983. Neural crest cells contribute to normal aorticopulmonary septation. Science 220: 1059. Lakkis M.M. and Epstein J.A. 1998. Neurofibromin modulation of ras activity is required for normal endocardial-mesenchymal transformation in the developing heart. Development 125: 4359. Le Douarin N. and Kalcheim C. 1999. The neural crest, 2nd edition. Cambridge University Press, Cambridge, United Kingdom. Li J., Chen F., and Epstein J.A. 2000. Neural crest expression of Cre recombinase directed by the proximal Pax3 promoter in transgenic mice. Genesis 26: 162. Li J., Liu K.C., Jin F., Lu M.M., and Epstein J.A. 1999. Transgenic rescue of congenital heart disease and spina bifida in Splotch mice. Development 126: 2495. Lindsay E.A., Botta A., Jurecic V., Carattini-Rivera S., Cheah Y.C., Rosenblatt H.M., Bradley A., and Baldini A. 1999. Congenital heart disease in mice deficient for the DiGeorge syndrome region. Nature 401: 379. Lindsay E.A., Vitelli F., Su H., Morishima M., Huynh T., Pramparo T., Jurecic V., Ogunrinu G., Sutherland H.F., Scambler

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P.J., Bradley A., and Baldini A. 2001. Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice. Nature 410: 97. Merscher S., Funke B., Epstein J.A., Heyer J., Puech A., Lu M.M., Xavier R.J., Demay M.B., Russell R.G., Factor S., Tokooya K., Jore B.S., Lopez M., Pandita R.K., Lia M., Carrion D., Xu H., Schorle H., Kobler J.B., Scambler P., Wynshaw-Boris A., Skoultchi A.I., Morrow B.E., and Kucherlapati R. 2001. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 104: 619. Moase C.E. and Trasler D.G. 1990. Delayed neural crest cell emigration from Sp and Spd mouse neural tube explants. Teratology 42: 171. _______ . 1991. N-CAM alterations in splotch neural tube defect mouse embryos. Development 113: 1049. _______ . 1992. Splotch locus mouse mutants: Models for neural

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Neural Crest Migration and Mouse Models of ...

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