Myocardin-Related Transcription Factor B Is Required in Cardiac Neural Crest for Smooth Muscle Differentiation and Cardiovascular Development

Home , Aorticopulmonary septum

Myocardin-related transcription factor B is required in cardiac neural crest for smooth muscle differentiation and cardiovascular development

Jian Li, Xiaohong Zhu, Mary Chen, Lan Cheng, Deying Zhou, Min Min Lu, Kevin Du, Jonathan A. Epstein, and Michael S. Parmacek*

Penn Cardiovascular Institute, Molecular Cardiology Research Center, University of Pennsylvania, Philadelphia, PA 19104

Communicated by Mark T. Keating, Harvard Medical School, Boston, MA, May 6, 2005 (received for review March 30, 2005) Members of the myocardin-related family of transcription factors ogies involving the cardiac outflow tract and great arteries that play critical roles in regulating vascular smooth muscle and cardiac are associated with common forms of congenital heart disease differentiation. To examine the function of myocardin-related observed in humans (5). transcription factor (MRTF)-B, mice were generated from ES cells A critical step in the formation of the cardiac outflow tract and harboring a conditional insertional mutation, or gene trap, of the of the great arteries is the differentiation of neural crest cells to MRTF-B gene. Expression of the MRTF-B mutant allele results in a specified SMCs. Relatively little is currently understood about fusion protein consisting of the N terminus of MRTF-B fused to the molecular mechanisms regulating differentiation of SMCs ␤-galactosidase, which is functionally null. Homozygous MRTF-B from neural crest progenitors. By contrast, SMCs arising from gene trap mice (MRTF-B؊/؊) die between embryonic day (E)17.5 lateral mesoderm and local mesenchyme are regulated by a and postnatal day 1 from cardiac outﬂow tract defects. MRTF-B is serum response factor (SRF)-dependent transcriptional regula- expressed in the premigratory neural crest, in rhombomeres 3 and tory program (6). SRF activates SMC transcription regulatory 5, and in the neural crest-derived mesenchyme surrounding the elements by physically associating with the SMC- and cardiac- aortic arch arteries. Consistent with the pattern of expression, restricted transcriptional coactivator myocardin (7–9). Mice E10.5 and E11.5 MRTF-B؊/؊ mutants exhibit deformation of aortic harboring a myocardin null mutation die at embryonic day arch arteries 3, 4, and 6 and severe attenuation of smooth muscle (E)10.5 and show no evidence of vascular SMC differentiation cell differentiation in the arch arteries and the aorticopulmonary in the dorsal aorta (10). It remains unclear whether identical, septum, despite normal migration and initial patterning of cardiac overlapping, or distinct transcriptional programs regulate dif- neural crest cells. Remarkably, the observed pathology was res- ferentiation of SMCs arising from the lateral mesoderm and cued and viable mice generated by intercrossing MRTF-B mutants cardiac neural crest. However, the finding that common forms of with mice expressing Cre recombinase under the transcriptional congenital heart disease involving neural crest-derived struc- control of the neural crest-restricted Wnt-1 promoter, which results tures are generally not associated with other vascular malfor- in restoration of normal MRTF-B expression in the neural crest. mations suggests that the developmental programs regulating Taken together, these studies reveal that MRTF-B plays a critical these processes are distinct. role in regulating differentiation of cardiac neural crest cells into Two myocardin-related transcription factors (MRTFs), des- smooth muscle and demonstrate that neural crest-derived smooth ignated MRTF-A and -B, have been described (11). Like myo- muscle differentiation is speciﬁcally required for normal cardio- cardin, these factors physically associate with SRF and activate vascular morphogenesis. SMC-restricted transcription. MRTF-A is expressed by multiple cell lineages and is a remarkably potent transcriptional coacti- congenital heart disease ͉ myocardin ͉ heart ͉ angiogenesis vator of some SRF-dependent genes (11–15). In response to RhoA-mediated and cytoskeletal signals, MRTF-A translocates he critical role that the cardiac neural crest plays in the from the cytoplasm to the nucleus and activates gene expression Tmorphogenetic program regulating cardiac outflow tract (12–14). By contrast, relatively little is currently understood development and formation of the great arteries has been about the function of MRTF-B, which is expressed in a unique revealed through classical anatomic neural crest ablation exper- developmentally regulated lineage-restricted fashion, including iments in combination with quail–chick cell transplantation expression in some SMCs and the heart (11). Compared with studies (1, 2). Recently, the application of genetic Cre-Lox myocardin and MRTF-A, the full length MRTF-B protein is a fate-mapping techniques and the generation of mouse models relatively weak transcriptional coactivator, although it contains recapitulating human congenital heart disease have begun to a powerful transcriptional activation domain (11). Of note, define the molecular basis of cardiac outflow tract development complete inhibition of RhoA-inducible SRF-mediated gene (3, 4). Neural crest cells arising in the dorsal neural tube between expression requires blockade of both MRTF-A and -B, suggest- the midotic placode and the third somite contribute to portions ing some redundancy of function between MRTF-A and -B (15). of the heart and great vessels leading to their designation as the Materials and Methods cardiac neural crest (1). In response to poorly understood Generation and Characterization of MRTF-B Gene Trap Mice. ES cells developmental cues, these cells migrate ventrally, populating the containing an insertional mutation or gene trap in the MRTF-B aortic arch arteries and cardiac outflow tract. The neural crest gene (clone RRJ478) were provided by BayGenomics, which can derivatives then differentiate into smooth muscle cells (SMCs) be accessed at http:͞͞baygenomics.ucsf.edu. The gene-trap vec- forming the tunica media of the great arteries and the ascending aorta (1). Concomitant with this migration, two advancing

columns of neural crest cells invade the cardiac outflow tract Abbreviations: SMCs, smooth muscle cells; SRF, serum response factor; En, embryonic day where they fuse, dividing the single great vessel, the truncus n; MRTF, myocardin-related transcription factor; Pn, postnatal day n. arteriosus, forming the aorticopulmonary septum and establish- *To whom correspondence should be addressed. E-mail: michael.parmacek@uphs. ing the aorta and pulmonary arteries (1). Alterations in this upenn.edu. complex developmental program lead to a spectrum of pathol- © 2005 by The National Academy of Sciences of the USA

8916–8921 ͉ PNAS ͉ June 21, 2005 ͉ vol. 102 ͉ no. 25 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503741102 Downloaded by guest on October 2, 2021 Fig. 1. Characterization of MRTF-B gene trap mice. (A) Schematic representation of the MRTF-B gene trap. (Top) The gene trap vector contains a splice acceptor (SA) sequence ﬂanked by loxP sites (triangles) subcloned 5Ј of the ␤geo cassette and a polyadenylation sequence (pA). (Middle) A partial restriction map of the mouse MRTF-B gene showing exon 10 and 11 sequences (rectangles). PCR primers are shown (arrows). (Bottom) A partial restriction map of the MRTF-B mutated allele. (B) Southern blot analysis of DNA prepared from the offspring of MRTF-Bϩ/Ϫ ϫ MRTF-Bϩ/Ϫ mating. The positions of the wild-type (9.8-kb) and mutant (6.5-kb) alleles are indicated to the left. (C) Northern blot analyses of MRTF-B gene expression in wild-type (ϩ͞ϩ), heterozygous (ϩ͞Ϫ), and null (Ϫ͞Ϫ) MRTF-B embryos. RNA was harvested from E12.5 embryos. The wild-type (9.5-kb) and mutant (7.5-kb) transcripts are shown to the left. (D) Real-time RT-PCR of MRTF-B gene expression in wild-type (ϩ͞ϩ), heterozygous (ϩ͞Ϫ), and null (Ϫ͞Ϫ) MRTF-B embryos. (E) MRTF-A- and –B-induced transactivation of the SM22␣ promoter in NIH 3T3 cells. NIH 3T3 cells were cotransfected with the indicated amounts (in micrograms) of expression plasmids and p-441SM22.luc. The data are presented as relative luciferase activities Ϯ SEM. (F) Forced expression of the MRTF-B⌬731 mutant protein does not repress myocardin-induced transactivation of the SM22␣ promoter in NIH 3T3 cells. NIH 3T3 cells were cotransfected with the indicated amounts (␮g) of expression plasmid and p-441SM22.luc.

tor (pGT0 lxf) contains a splice-acceptor sequence flanked by described previously (9, 13). NIH 3T3 cells were cotransfected loxP sites (floxed) subcloned 5Ј of the ␤geo reporter cassette with 200 ng of the indicated luciferase reporter plasmid, 100–500 encoding a fusion protein consisting of the bacterial lacZ gene ng of the indicated expression plasmid, and 10 ng of the fused to neomycin phosphotransferase II (16). DNA sequence phRL-TK (-Int) reference plasmid (Promega), as described (18). analysis revealed that the floxed splice acceptor and ␤-geo cassette integrated 191 bp 3Ј of MRTF-B exon 10 generating Immunohistochemistry and in Situ Hybridization Analyses. Immuno- truncated MRTF-B lacking a transcriptional activation domain histochemistry and in situ hybridization protocols are available (Fig. 1A). Clone RRJ478 ES cells were injected into blastocysts at www.uphs.upenn.edu͞mcrc͞histology͞histologyhome.html from C57BL͞6J mice, and mutant mice were generated and (19). Antibodies included: anti-MF20 monoclonal antibody (De- characterized as described (17). To rescue MRTF-BϪ/Ϫ mice velopmental Studies Hybridoma Bank, Iowa City, IA); anti- specifically in neural crest derivatives, MRTF-Bϩ/Ϫ mice were smooth muscle ␣-actin monoclonal antibody (Dako); neurofila- intercrossed withWnt-1-Cre transgenic mice kindly provided by ment 2H3 monoclonal antibody (Developmental Studies A. McMahon (Harvard University, Boston) (3). Hybridoma Bank); anti-Krox20 polyclonal antibody (Covance Research Products, Denver, PA). The plexinA2 antisense cRNA Northern Blot and Real-Time RT-PCR Analyses. Northern blot analysis probe was described previously (20). was performed as described (9). The relative intensity of the 9.5-kb MRTF-B and 7.5-kb mutant MRTF-B transcripts was quantified by Intracardiac Ink Injections. India ink was injected intracardially or using a Molecular Dynamics PhosphorImager. Real-time RT-PCR into the descending aorta of mouse embryos by using a pulled BIOLOGY

was performed and quantified as described (9). glass pipette and immediately photographed as described (21). DEVELOPMENTAL

Plasmids and Transient Cotransfection Analyses. The pcDNA- Results MRTF-B and pcDNA-MRTF-B⌬731 expression plasmids en- Generation of MRTF-B Gene Trap Mice. Mice were generated from ES code full-length mouse MRTF-B and the N-terminal 731 amino cells containing an insertional mutation or ‘‘gene trap’’ in intron 10 acids of mouse MRTF-B (NM࿝153588), respectively. pcDNA3.1- of the mouse MRTF-B gene. As shown in Fig. 1A, gene trap vectors myocardin, pcDNA3-MRTF-A, and the pcDNA-Myocar- were designed to generate spliced fusion transcripts between a din⌬585 expression plasmids and the p-441SM22.luc, pPI- reporter gene (␤geo) and the endogenous gene (MRTF-B) present Act.luc, and pPI-Myo.luc luciferase reporter plasmids were at the site of integration (16, 22). In this gene trap vector, the splice

Table 1. Genotype distribution of MRTF-B embryonic and perinatal lethality Genotype E12.5 (53) E13.5 (40) E14.5 (66) E16.5 (142) E17.5 (55) E18.5 (113) P0 (294) P1 (281)

ϩ͞ϩ 17 12 17 39 14 36 87 87 ϩ͞Ϫ 24 19 35 72 32 58 190 190 Ϫ͞Ϫ 12 (23%) 9 (23%) 14 (21%) 31 (22%) 9 (16%) 18 (16%) 17 (6%) 4 (1.4%)

Li et al. PNAS ͉ June 21, 2005 ͉ vol. 102 ͉ no. 25 ͉ 8917 Downloaded by guest on October 2, 2021 Table 2. Summary of phenotypes in MRTF-B-deﬁcient embryos expression plasmids encoding MRTF-A and -B, respectively, and Ventricular septal defect 16͞16 the SMC-restricted p-441SM22.luc reporter plasmid (Fig. 1E). ⌬ Persistent truncus ateriosus 7͞16 In contrast, forced expression of the mutant MRTFB 731 ␣ Double-outlet right ventricle 8͞16 protein failed to transactivate the SMC-restricted SM22 pro- Right-sided aortic arch 1͞16 moter (Fig. 1E). As expected, forced expression of a dominant Interrupted aortic arch 2͞16 negative myocardin mutant protein (MYCD⌬585) suppressed Perivascular hemorrhage and edema 6͞16 myocardin-induced SM22␣ promoter activity (Fig. 1F). By contrast, forced expression of MRTFB⌬731 failed to suppress myocardin- or MRTF-A-induced SM22␣ promoter activity (Fig. acceptor is flanked by LoxP sites (floxed) to facilitate Cre-mediated 1F and data not shown). These data demonstrate that the mutant cell lineage-restricted rescue of the mutant allele (16). Southern MRTF-B allele is functionally null as a transcriptional coactivator blot analyses confirmed transmission of the targeted allele contain- and does not function as a dominant negative to repress activity ing the MRTF-B gene trap through the germline (Fig. 1B). Northern of myocardin or MRTF-A. blot analyses demonstrated expression of both the wild-type MRTF-Bϩ/ϩ lacZ Characterization of MRTF-B-Deficient Embryos. Intercrossing MRTF- MRTF-B transcript ( ) and the mutated MRTF-B- ϩ/Ϫ fusion transcript (MRTF-Bϩ/Ϫ) (Fig. 1C). The native 9.5-kb B heterozygotes failed to generate the anticipated Mendelian MRTF-BϪ/Ϫ pattern of inheritance (Table 1). Of 281 postnatal day (P)1 MRTF-B transcript was also detectable in mice dem- Ϫ/Ϫ onstrating low-level alternative splicing around the insertional gene offspring analyzed, only 4 (1.4%) null (MRTF-B ) mice were trap mutation (Fig. 1C). Quantification of the hybridization signals identified. At birth (P0), 6% of 294 pups were genotyped as null, 65% were heterozygotes (MRTF-Bϩ/Ϫ), and 30% were wild-type revealed wild-type MRTF-B mRNA represented 5% of total ϩ/ϩ MRTF-B transcripts in MRTF-BϪ/Ϫ embryos. This finding was (MRTF-B ). Through E16.5, the percentage of MRTF-B null confirmed by real-time RT-PCR (Fig. 1D). Thus, the phenotype of mice varied between 21% and 23%, suggesting that MRTF-B MRTF-BϪ/Ϫ mice may represent a weak hypomorphic rather than null mice are generally viable until late in gestation. However, at true null phenotype. E17.5 and E18.5, only 16% of embryos were MRTF-B null. Consistent with prior studies (11), inductions in luciferase These data demonstrate that, in contrast to myocardin null activity were observed in NIH 3T3 cells cotransfected with embryos that die at E10.5 (10), MRTF-B null embryos develop

Fig. 3. MRTF-B is expressed in the cardiac neural crest and aortic arch arteries during embryonic development. (A) An E8.5 MRTF-Bϩ/Ϫ embryo demonstrat- Fig. 2. Cardiovascular abnormalities in MRTF-B null mice. (A) Wild-type E18.5 ing expression of the MRTF-B-lacZ fusion protein (arrows) in rhombomeres 3 embryo demonstrating left-sided aortic arch (arrow). (B) MRTF-BϪ/Ϫ embryo and 5 of the dorsal neural folds. (B) An E8.5 embryo demonstrating Krox20 with interrupted aortic arch (arrow). (C) MRTF-BϪ/Ϫ embryo with right-sided expression (arrows) in rhombomeres 3 and 5. (C) An E9.5 MRTF-Bϩ/Ϫ embryo aortic arch (arrow). (D–F) Rostral (D) to caudal (F) sections demonstrating the demonstrating lacZ-positive cells populating the aorta (arrows), aortic arch aorta (Ao) arising from the LV and the pulmonary artery (PA) arising from the arteries, cardiac outﬂow tract, and heart. (D) An E11.5 MRTF-Bϩ/Ϫ embryo RV in a control E16.5 embryo. (G–I) Double-outlet right ventricle in an E16.5 demonstrating lacZ expression in the cells populating the mesenchyme sur- MRTF-BϪ/Ϫ mutant embryo shown rostral (G) to caudal (I). Both the aorta (Ao) rounding aortic arch arteries 3, 4, and 6 (arrows). (E and F) Higher-power view and pulmonary artery (PA) arise from the RV, and there is an obligate ven- of D demonstrating colocalization of ␤-galactosidase activity (blue in E) and tricular septal defect (VSD). (J–L) Truncus arteriosus defect in an E16.5 MRTF- SM-␣-actin (red in F) in the cells populating the third left aortic arch artery. BϪ/Ϫ mutant embryo shown rostral (J) to caudal (L). (E Inset) Colocalization of lacZ (blue) and SM-␣-actin (orange) expression.

8918 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503741102 Li et al. Downloaded by guest on October 2, 2021 Fig. 4. Cardiac neural crest cells migrate appropriately but exhibit a block in SMC differentiation in MRTF-BϪ/Ϫ embryos. (A–D) Intracardiac ink injection to visualize the pharyngeal arch arteries in E10.5 (A and B) and 11.5 (C and D) wild-type (A and C) and MRTF-BϪ/Ϫ (B and D) embryos. Note regression of aortic arch arteries 4 and 6 in MRTF-BϪ/Ϫ mutant embryos (B and D). (E–H) Coronal sections of E11.5 wild-type (E and F) and MRTF-BϪ/Ϫ (G and H) embryos demonstrate expression of SM-␣-actin (E) and plexinA2 (F) in the mesenchyme surrounding the aortic arch arteries in wild-type embryos. By contrast, in E11.5 MRTF-B-deficient embryos (G and H), expression of SM-␣-actin is markedly down-regulated in the aortic arch arteries (G, arrows). In this representative embryo, expression of SM-␣-actin in the fourth and sixth right arch arteries was barely detectable (G). Expression of plexinA2 was readily detectable in the pharyngeal mesenchyme surrounding the aortic arch arteries (H). PlexinA2 gene expression in wild-type and MRTF-BϪ/Ϫ embryos in the pharyngeal mesenchyme was comparable (compare F and H). (I–L) Sagittal sections cut at the level of the aorticopulmonary septum of E11.5 wild-type (I and J) and MRTF-BϪ/Ϫ (K and L) embryos demonstrating coexpression of SM-␣-actin (I) and plexinA2 (J) in the nascent aorticopulmonary septum of wild-type embryos (arrows, I and J). By contrast, in MRTF-BϪ/Ϫ embryos, the aorticopulmonary septum fails to develop, and expression of SM-␣-actin is severely down-regulated at the level of the outflow tract (arrows in K). PlexinA2-expressing cells (arrows in L) are observed, demonstrating that neural crest cells migrated appropriately to the cardiac outflow tract.

a vasculature that is capable of sustaining an embryo through Bϩ/Ϫ embryos. Surprisingly, at E8.5, intense ␤-galactosidase late gestation, and that perinatal demise initiates at about E16.5. activity was observed in the dorsal neural folds at the level of To determine the cause of demise of MRTF-B null embryos, rhombomeres 3 and 5 (Fig. 3A). This lacZ activity colocalized MRTF-BϪ/Ϫ embryos were characterized at E16.5 and E18.5 with cells expressing Krox20, a marker of neural crest (Fig. 3B) (Table 2). The embryos did not appear to be growth retarded, (23). Intense blue staining was also observed throughout the although 6 of 16 MRTF-B null embryos appeared to be grossly heart at E8.5 and E9.5 (Fig. 3A). LacZ expression surrounding edematous. Inspection of the heart and great arteries revealed the aortic arch arteries and the thoracic aorta was still evident at cardiac outflow tract defects in all 16 MRTF-BϪ/Ϫ embryos E10.5 (Fig. 3C). At E11.5, lacZ-positive MRTF-B-expressing BIOLOGY analyzed (Fig. 2). Eight of 16 embryos exhibited double-outlet cells populated branchial arch arteries 3, 4, and 6 (Fig. 3D, DEVELOPMENTAL right ventricle (Fig. 2 G–I), and 7 of 16 embryos exhibited arrows). MRTF-B staining colocalized with expression of SM- persistent truncus arteriosus (Fig. 2 J–L). In addition, two ␣-actin in the aortic arch arteries (Fig. 3 E and F). The finding mutant embryos exhibited an interrupted aortic arch (arrow, Fig. that MRTF-B is expressed at high levels in the dorsal neural tube 2B), and one exhibited a right-sided aortic arch (arrow, Fig. 2C). and subsequently in the aortic arch arteries suggested that Large ventricular septal defects were observed in all MRTF-B expression of MRTF-B in the cardiac neural crest could be null embryos but were never observed in wild-type embryos after responsible for the outflow tract pathology observed in MRTF-B E14.5 (Fig. 2 F and I). Complex structural heart disease in null embryos. association with signs of congestive heart failure (edema) sug- To examine the timing and origin of the cardiac outflow tract gests that prenatal lethality in some embryos is due to cardio- defects, India ink was injected into the heart and dorsal aorta of vascular insufficiency. Structural heart defects such as those wild-type (Fig. 4 A and C) and MRTF-BϪ/Ϫ (Fig. 4 B and D) observed in MRTF-B null pups are sufficient to account for embryos harvested at E10.5 and E11.5. In wild-type embryos, postnatal lethality of the remaining animals. three right-sided and three left-sided arch arteries were clearly delineated (arrows, Fig. 4 A and C). By contrast, generally only MRTF-B Expression in the Cardiac Neural Crest and Aortic Arch Artery one or two right- and left-sided aortic arch arteries were Malformations. The pattern of MRTF-B gene expression was observed in MRTF-B-deficient embryos (arrows, Fig. 4 B and mapped by characterizing the pattern of lacZ staining in MRTF- D). Several different abnormal arch artery patterns were ob-

Li et al. PNAS ͉ June 21, 2005 ͉ vol. 102 ͉ no. 25 ͉ 8919 Downloaded by guest on October 2, 2021 served in MRTF-B null embryos. In some cases, the left fourth arch artery, which gives rise to the definitive aortic arch, was absent, consistent with our observation of interrupted aortic arch phenotypes in some MRTF-B null animals. In other embryos, one or both sixth arch arteries, which normally give rise to the pulmonary trunk and ductus arteriosus, were not visual- ized. These data suggest that abnormal aortic arch artery patterning is evident as early as E10.5 in a manner consistent with subsequent structural heart disease seen in newborn MRTF-B-deficient pups. At E11.5, SM-␣-actin, an early SMC marker, is expressed strongly in the aortic arch arteries of wild-type mice (Fig. 4E, arrows). Consistent with their neural crest origin, these cells (arrows) also coexpress the neural crest marker, plexinA2 (Fig. 4F). By contrast, E11.5 MRTF-B-deficient embryos exhibited multiple defects in aortic arch patterning, including severely dilated aortic arch arteries, absent arch arteries, and dilated aortic sac (Fig. 4G). Moreover, SM-␣-actin was absent or severely attenuated in the aortic arch arteries of MRTF-B null embryos (arrow, Fig. 4G). No obvious difference in the expression pattern of plexinA2 was observed in the mesenchyme surrounding aortic arch arteries of wild-type and MRTF-B null embryos (compare Fig. 4 F and H). In addition at E11.5, SM-␣-actin-positive cells may be ob- Fig. 5. Neural crest-restricted rescue of MRTF-B gene trap mice. (A) served forming the nascent septum that divides the truncus Schematic representation of the MRTF-B gene trap rescue strategy. In Ϫ Ϫ ϩ arteriosus into the aorta and pulmonary artery (Fig. 4I). These MRTF-B / ͞Cre mice, the splice acceptor sequence (SA) is deleted, regener- cells express the plexinA2 gene, confirming their origin as neural ating the native MRTF-B transcript speciﬁcally in neural crest cells. (B and C) Ϫ/Ϫ͞ Ϫ Ϫ/Ϫ͞ ϩ crest (Fig. 4J). By contrast, in MRTF-B null embryos, the LacZ expression (blue staining) in MRTF-B Cre (B) and MRTF-B Cre (C) E11.5 embryos. Note the failure of the neural crest-derived branchial arch aorticopulmonary septum was either absent or severely dimin- region of the Creϩ embryo to stain blue (arrows). ished (arrows), and SM-␣-actin-positive cells were rarely observed (arrows, Fig. 4K). However, two columns of plexinA2- expressing cells were readily observed populating the cardiac Discussion outflow tract at the level of the aorticopulmonary septum The observed phenotype of MRTF-B-deficient embryos dem- (arrows, Fig. 4L), demonstrating that the cardiac neural crest onstrates that, despite the high-level sequence identity between cells migrated appropriately to the cardiac outflow tract. Taken MRTFs and the capacity of each MRTF to transactivate multiple together, these data reveal a specific block in the capacity of SMC-restricted transcriptional elements (11), the function of cardiac neural crest derivatives to undergo SMC differentiation MRTF-B in the cardiac neural crest is unique and nonredundant in MRTF-B-deficient mice. with other MRTF family members. MRTF-B-deficient embryos survived only through late gestation and demonstrated cell Neural Crest-Specific Rescue of MRTF-B Null Embryos. To confirm that autonomous defects in neural crest differentiation into SMCs the outflow tract defects and mortality observed in MRTF-B- cells accompanied by cardiac outflow tract defects. The conclu- deficient mice resulted from a cell autonomous defect of cardiac sion that the primary defect in MRTF-B-deficient mice is a cell ϩ Ϫ neural crest, MRTF-B / mice were intercrossed with mice express- autonomous defect in cardiac neural crest derivatives rather than ing Cre under the transcriptional control of the neural crest- a primary defect in cardiac morphogenesis and͞or myocyte restricted Wnt-1 promoter (Fig. 5A) (3). To confirm that Wnt1-Cre differentiation is substantiated by the finding of normal heart ϩ (Wnt1-Cre ) mice efficiently excised the conditional MRTF-B morphology in rescued MRTF-BϪ/Ϫ͞Wnt-1-Creϩ mice. As such, Ϫ Ϫ gene trap, expression of ␤-galactosidase in MRTF-B / ͞Wnt1- these data serve to identify MRTF-B as a candidate gene for ϩ Ϫ Ϫ Ϫ Cre and MRTF-B / ͞Wnt1-Cre mice was compared. In E11.5 common forms of congenital heart disease, including persistent embryos stained for extended periods of time, ␤-galactosidase truncus arteriosus, interrupted aortic arch, and double-outlet ϩ Ϫ expression was virtually identical in Cre and Cre embryos, except right ventricle. in the neural crest-derived regions of mandible, pharyngeal arches, It is likely that the respective functions of MRTF-B and and cardiac outflow tract (arrows, Fig. 5 B and C). Most impor- myocardin during embryonic development are in large part ϩ Ϫ tantly, of 74 live-born offspring generated from MRTF-B / ͞Wnt1- related to differences in their temporal and spatial patterns of ϩ ϩ Ϫ ϩ Cre ϫ MRTF-B / ͞Wnt-1-Cre intercrosses, 15 (20.2%) MRTF- expression. MRTF-B, but not myocardin, is expressed in the Ϫ Ϫ ϩ B / ͞Wnt1-Cre offspring were observed at P1 compared with two cardiac neural crest as early as E8.5, and the MRTF-B gene of 74 (2.7%) offspring of the MRTF-BϪ/Ϫ͞Wnt1-CreϪ genotype. continues to be expressed in the migrating cardiac neural crest All MRTF-BϪ/Ϫ͞Wnt1-Creϩ E16.5–18.5 embryos and P14 mice at least through E10.5. In addition, MRTF-B is expressed in the appeared phenotypically normal, and outflow tract defects and embryonic heart and lateral mesoderm-derived vasculature but, ventricular septal defects were not observed. Furthermore, 100% other than secondary heart defects that presumably were related (15͞15) of the MRTF-BϪ/Ϫ͞Wnt1-Creϩ mice were alive and well 12 to the outflow tract defects, these tissues appeared grossly weeks after birth, whereas only two MRTF-BϪ/Ϫ͞Wnt1-CreϪ lit- normal in MRTF-B-deficient mice. Therefore, MRTF-B is not termates survived past P1. Hence, cardiac outflow tract defects in required for expression of differentiated SMC markers in the MRTF-B null mice are efficiently rescued by neural crest-restricted heart and peripheral vasculature during embryonic develop- expression of Cre, supporting the conclusion that expression of ment, although it may mediate redundant functions with MRTF-B in the cardiac neural crest is required for differentiation MRTF-A and͞or myocardin in these tissues. By contrast, despite of neural crest derivatives to SMCs and for cardiac outflow tract the fact that myocardin and MRTF-B are coexpressed in em- patterning. bryonic vascular SMCs, myocardin null mice die at E10.5, and

8920 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503741102 Li et al. Downloaded by guest on October 2, 2021 differentiated SMCs are not observed in the dorsal aorta (12). Analysis of the MRTF-B-deficient mice suggests a molecular Thus, MRTF-B cannot compensate for loss of myocardin at this model in which MRTF-B sits at a convergence point in cardiac early stage of embryonic angiogenesis in these cells. neural crest, integrating multiple extracellular signals and de- Formation of the cardiac outflow tract and great arteries is velopmental cues ultimately promoting vascular SMC differen- controlled by multiple distinct signals originating in the pharyn- tiation. This hypothesis is supported by the finding that MRTF-B geal endoderm and local mesoderm that converge upon the is expressed at high levels in the cardiac neural crest, and cardiac neural crest cells acting in a combinatorial fashion (1, 2, MRTF-B-deficient embryos recapitulate the outflow tract de- 24). Neural crest-specific inactivation of the type 1 bone mor- fects observed in bone morphogenetic protein͞semaphorin͞ phogenetic protein receptor Alk2 causes a failure of outflow VEGF mutant mice (21, 26, 27). In this regard, it is noteworthy tract septation with deficient differentiation of neural crest- that each of these growth factors is secreted by cells in the derived SMCs (21). Inactivation of Semaphorin 3C, a secreted pharyngeal endoderm and mesoderm surrounding the aortic class 3 semaphorin, results in interruption of the aortic arch and arch arteries and outflow tract (27). We postulate that the persistent truncus arteriosus (25). Class 3 semaphorins bind to appropriate combination of signals promotes the translocation heterodimeric receptors composed of neuropilin and plexin subunits. Neuropilin 1-deficient mice display persistent truncus of MRTF-B from the cytoplasm to the nucleus, where it phys- arteriosus, and both neuropilin 1 and plexinA2 are expressed by ically associates with SRF-activating transcription of a set of migrating cardiac neural crest cells (20, 26). However, interpre- genes promoting SMC differentiation (14). The demonstration tation of these data is complicated, because neuropilin receptors that forced expression of MRTF-B and SRF transactivates also bind to VEGF. Selective ablation of the VEGF-165 isoform, multiple SMC-transcriptional regulatory elements in non-SMCs which signals via neuropilin-containing receptors, results in a and that both MRTF-B and SRF are required to activate spectrum of outflow tract defects recapitulating DiGeorge syn- endogenous SMC genes in SRF-deficient ES cells (J.L., unpub- drome (27). Although neural crest-derived SMC differentiation lished observation) strongly supports the contention that has been noted to be abnormal in several mouse models with MRTF-B acts as a critical transcriptional coactivator of SRF in outflow tract defects, it has not been clear whether this repre- the cardiac neural crest. As such, MRTF-B mediates a cell sents the primary cause of the morphogenetic abnormalities. autonomous function in the cardiac neural crest integrating However, loss of MRTF-B is likely to represent a specific loss of multiple signals that regulate the process of neural crest differ- SMC differentiation due to deficient activation of SMC struc- entiation into SMCs and the morphogenetic program controlling tural gene promoters in neural crest cells. Hence, the develop- formation of the cardiac outflow tract and great arteries. ment of typical forms of congenital heart disease in MRTF-B mutants provides compelling evidence that disruption of SMC This work was supported in part by National Institutes of Health differentiation is sufficient to produce cardiac outflow tract Grants RO1-HL56915 and PO1-HL075380 (to M.S.P.) and PO1- defects. HL075380 (to J.A.E.).

1. Kirby, M. L., Gale, T. F. & Stewart, D. E. (1983) Science 220, 1059–1061. 15. Cen, B., Selvaraj, A., Burgess, R. C., Hitzler, J. K., Ma, Z., Morris, S. W. & 2. Creazzo, T. L., Godt, R. E., Leatherbury, L., Conway, S. J. & Kirby, M. L. Prywes, R. (2003) Mol. Cell. Biol. 23, 6597–6608. (1998) Annu. Rev. Physiol. 60, 267–286. 16. Skarnes, W. C., Auerbach, B. A. & Joyner, A. L. (1992) Genes Dev. 6, 903–918. 3. Jiang, X., Rowitch, D. H., Soriano, P., McMahon, A. P. & Sucov, H. M. (2000) 17. Morrisey, E. E., Tang, Z., Sigrist, K., Lu, M. M., Jiang, F., Ip, H. S. & Parmacek, Development (Cambridge, U.K.) 127, 1607–1616. M. S. (1998) Genes Dev. 12, 3579–3590. 4. Li, J., Chen, F. & Epstein, J. A. (2000) Genesis 26, 162–164. 18. Solway, J., Seltzer, J., Samaha, F. F., Kim, S., Alger, L. E., Niu, Q., Morrisey, 5. Gruber, P. J. & Epstein, J. A. (2004) Circ. Res. 94, 273–283. E. E., Ip, H. S. & Parmacek, M. S. (1995) J. Biol. Chem. 270, 13460–13469. 6. Owens, G. K., Kumar, M. S. & Wamhoff, B. R. (2004) Physiol. Rev. 84, 767–801. 19. Morrisey, E. E., Ip, H. S., Lu, M. M. & Parmacek, M. S. (1996) Dev. Biol. 177, 7. Wang, D., Chang, P. S., Wang, Z., Sutherland, L., Richardson, J. A., Small, E., 309–322. Krieg, P. A. & Olson, E. N. (2001) Cell 105, 851–862. 20. Brown, C. B., Feiner, L., Lu, M. M., Li, J., Ma, X., Webber, A. L., Jia, L., Raper, 8. Wang, D. Z. & Olson, E. N. (2004) Curr. Opin. Genet. Dev. 14, 558–566. J. A. & Epstein, J. A. (2001) Development (Cambridge, U.K.) 128, 3071–3080. 9. Du, K. L., Ip, H. S., Li, J., Chen, M., Dandre, F., Yu, W., Lu, M. M., Owens, 21. Kaartinen, V., Dudas, M., Nagy, A., Sridurongrit, S., Lu, M. M. & Epstein, J. A. G. K. & Parmacek, M. S. (2003) Mol. Cell. Biol. 23, 2425–2437. (2004) Development (Cambridge, U.K.) 131, 3481–3490. 10. Li, S., Wang, D. Z., Wang, Z., Richardson, J. A. & Olson, E. N. (2003) Proc. 22. Gossler, A., Joyner, A. L., Rossant, J. & Skarnes, W. C. (1989) Science 244, 463–465. Natl. Acad. Sci. USA 100, 9366–9370.. 23. Ghislain, J., Desmarquet-Trin-Dinh, C., Gilardi-Hebenstreit, P., Charnay, P. & 11. Wang, D. Z., Li, S., Hockemeyer, D., Sutherland, L., Wang, Z., Schratt, G., Frain, M. (2003) Development (Cambridge, U.K.) 130, 941–953. Richardson, J. A., Nordheim, A. & Olson, E. N. (2002) Proc. Natl. Acad. Sci. 24. Kirby, M. L. & Waldo, K. L. (1995) Circ. Res. 77, 211–215. USA 99, 14855–14860. 25. Feiner, L., Webber, A. L., Brown, C. B., Lu, M. M., Jia, L., Feinstein, P., 12. Miralles, F., Posern, G., Zaromytidou, A. I. & Treisman, R. (2003) Cell 113, Mombaerts, P., Epstein, J. A. & Raper, J. A. (2001) Development (Cambridge, 329–342. U.K.) 128, 3061–3070. BIOLOGY 13. Du, K. L., Chen, M., Li, J., Lepore, J. J., Mericko, P. & Parmacek, M. S. (2004) 26. Kawasaki, T., Kitsukawa, T., Bekku, Y., Matsuda, Y., Sanbo, M., Yagi, T. & J. Biol. Chem. 279, 17578–17586. Fujisawa, H. (1999) Development (Cambridge, U.K.) 126, 4895–4902. DEVELOPMENTAL 14. Kuwahara, K., Barrientos, T., Pipes, G. C., Li, S. & Olson, E. N. (2005) Mol. 27. Stalmans, I., Lambrechts, D., De Smet, F., Jansen, S., Wang, J., Maity, S., Kneer, Cell. Biol. 25, 3173–3181. P., von der Ohe, M., Swillen, A., Maes, C., et al. (2003) Nat. Med. 9, 173–182.

Li et al. PNAS ͉ June 21, 2005 ͉ vol. 102 ͉ no. 25 ͉ 8921 Downloaded by guest on October 2, 2021