Neural Crest Cells Retain Multipotential Characteristics in the Developing Valves and Label the Cardiac Conduction System
http://www.100md.com
Tomoki Nakamura, Melissa C. Colbert, Jef
参见附件。
the Department of Pediatrics, Division of Molecular Cardiovascular Biology, Children’s Hospital Research Foundation, Cincinnati, Ohio.
Abstract
Multipotent neural crest cells (NCCs) are a major extracardiac component of cardiovascular development. Although recognized as contributing cells to the arterial valves at early developmental stages, NCC persistence in the valves at later times or in the adult heart is controversial. We analyzed NCC persistence and contributions to both semilunar and atrioventricular (AV) valves in the mature heart. Two NCC-specific promoters driving Cre recombinase, Wnt1-Cre and P0-Cre, were mated with floxed reporter mice, R26R or CAG-CAT-EGFP, to map NCC fate. Hearts were analyzed before aorticopulmonary (AP) septation through adult stages. As previously demonstrated, strong NCC labeling was detected in ventral and dorsal outflow cushions before AP septation. In contrast to previous reports, we found that substantial numbers of labeled cells persisted in the semilunar valves in late fetal, neonatal, and adult hearts. Furthermore, NCCs were also found in the AV valves, almost exclusively in the septal leaflets. NCCs in the AV valves expressed melanocytic and neurogenic markers. However, cells labeled in the proximal cardiac conduction system exhibited neurogenic and gliagenic markers, whereas some NCCs expressed no differentiation specific markers. These results suggest that cardiac NCCs contribute to the mature valves and the cardiac conduction system and retain multipotent characteristics late in development.
Key Words: development heart valves embryonic development transgenic mice genetics
Introduction
The importance of the neural crest contribution to cardiovascular development has been recognized for more than 20 years.1,2 Neural crest cells (NCCs) provide a major extracardiac source of mesenchyme to the outflow tract (OFT) and postganglionic neural progenitors that innervate the heart. Seminal work using NCC extirpation and analysis of quail chick chimeras demonstrated that NCCs contributed to the aorticopulmonary (AP) septum, the tunica media of the great arteries, the OFT septum, and the semilunar valves. However, crest-derived cells were seldom found beyond the atrioventricular (AV) canal or in the AV valves and their association with the cardiac conduction system (CCS) remains controversial.
The fate of these unique migratory, multipotential cells has been studied using extrinsic and intrinsic marking methods to facilitate mapping their developmental progress.3 Most of the early fate-mapping analyses were performed using avian or amphibian chimeric embryos, with more recent studies using retroviral labeling in chick embryos.4 Fukiishi and Morriss-Kay were the first to use vital dye methods to follow cranial NCC migration in mammals.5 In recent years, promoter–reporter constructs6 or double-transgenic analysis7 using lacZ or GFP reporter mice and neural crest–specific Cre recombinase has facilitated genetic marking of neural crest in mice. Several different transgenic lines carry neural crest–specific Cre recombinase activity, including Wnt1,8 P0,9 Pax3,10,11 and HtPA.12
These model systems have provided new data on neural crest behavior in mice but have also caused some confusion. Jiang et al13 established a standard for following neural crest–derived cells during cardiovascular development, demonstrating that -gal expression in Wnt1-Cre::R26R14–positive embryos reproduced or expanded most of the patterns of neural crest–invested tissues and structures described in quail chick chimeras.6 Subsequently, other investigators used this double transgenic model to follow NCC migration into the heart15 or analyze cell lineage in cardiac valves.16 Other models of NCC-restricted recombination using P0, Pax3, or HtPA with lacZ-based reporter mice show consistent major crest contribution to the great vessels, OFT septum and semilunar valves.9–12 Most differences among the data occur in the quantitative levels of recombination or perdurance of expression. For example, the number or intensity of labeled cells in the aortic valves range from insubstantial using Wnt1-Cre recombination13 to significant cell labeling in the aortic and pulmonary valves, as reported in HtPA-Cre recombined mice.12 However qualitative differences in expression were noted in the same model of recombination. Poelmann et al15 briefly described contiguous -gal expression in the proximal conduction system and bundle branches in Wnt1-Cre::R26R embryos that was not described by Jiang et al.13 Therefore, inconsistencies in the developmental expression patterns of neural crest–derived cells exist among models, even when the same recombination system is used.
In this report, we revisit neural crest lineage tracing, using 2 well-characterized neural crest–specific promoters for recombination, Wnt1-Cre8 and P0-Cre,9 and 2 distinct reporter mice, R26R for -gal expression14 and a floxed enhanced green fluorescent protein (EGFP) reporter, to examine crest in situ. This approach facilitated direct comparison between these models at similarly staged developmental times. Furthermore, we bred both recombinase and reporter mice onto a homogeneous C57BL/6J background to eliminate any genetic variation in Cre activity, the behavior of NCCs or reporter expression. As the data are consistent among most models at early developmental stages through midgestation, we concentrated our analyses on the distribution of neural crest–derived cells during later gestation–neonatal stages and in adult hearts. In contrast to prior reports, we see NCCs in AV valves and in late stage expression in the maturing CCS. Although some neural crest–derived cells exhibit melanocytic, neurogenic, and gliagenic characteristics, not all are stained with specific markers, suggesting some may contribute to the newly described neural crest stem cell population in the adult heart.17
Materials and Methods
Transgenic Animals
Animal use was in accordance with Institutional Guidelines with all experiments reviewed and approved by the Institutional Animal Care Committee. Mice were obtained either from our preexisting colonies or, for the PO-Cre animals, From Erik Meyers, Duke University. Fate mapping used a Cre-loxP recombination strategy from embryonic day 11.5 (E11.5) through 8 weeks postnatally. Wnt1-Cre8 and Rosa26 (R26R)14 mice were bred onto the C57BL/6J background for more than 10 generations as were the other lines used in these studies. Wnt1-Cre and P0-Cre, which specifically label NCCs and their derivatives, were mated with either R26R or CAG-CAT-EGFP reporter mice. The CAG-CAT-EGFP reporter construct was generated by inserting a 3.2kb SalI–BamHI fragment containing the CMV- actin promoter and loxP flanked CAT gene18 upstream of the enhanced green fluorescent protein (EGFP) cassette. Timed pregnancies were established with the morning of a vaginal mucous plug defined as E0.5.
-Gal Expression
The Wnt1-Cre::R26R and P0-Cre::R26R mice were prepared and processed for -gal expression as described19 with the following modifications. The thoracic viscera were removed and drop fixed in 0°C 2% paraformaldehyde, 0.2% glutaraldehyde in PBS (pH 7.4). Tissues were washed extensively with PBS and immersed in a PBS/substrate solution containing 2 mmol/L MgCl2, 5 mmol/L potassium ferricyanide, 5 mmol/L ferrocyanide, and 0.1% X-gal dissolved in dimethyl formamide. For frozen sections, tissues were postfixed with 4% paraformaldehyde in PBS, infiltrated with sucrose, and embedded in OCT. Serial 8-μm sections were cut for embryos younger than E13.5 and 10-μm sections for mice older than E14.5. The samples were counterstained with nuclear fast red.
Immunohistochemistry
Wnt1-Cre::EGFP hearts were fixed overnight in 0°C 4% paraformaldehyde in PBS and prepared for immunohistochemistry as described.20 Primary antibodies used were: (1) rat anti-CD31 (1:100 dilution, BD biosciences) to detect endothelial cells; (2) goat anti–procollagen type I (1:100 dilution, Santa Cruz Biotechnology Inc) to identify fibroblasts; (3) goat anti–collagen 1 type II (1:100 dilution, Santa Cruz Biotechnology Inc) to identify chondrocytes; (4) rabbit anti–cleaved caspase 3 (1:100 dilution, Cell Signaling Technology) to detect apoptotic cells; (5) rabbit anti–tyrosinase-related protein 1 (TRP1) (1:50 dilution, Santa Cruz Biotechnology Inc) to identify melanocytes and melanocytic precursors; (6) rabbit anti–neuronal class III -tubulin (TUJ1) (1:2000 dilution, Covance) to identify neurogenic cells; (7) rabbit anti–brain lipid–binding protein (BLBP) (1:1000 dilution), which identifies radial glia.21 Alexa Fluor 568 (donkey anti-goat IgG for procollagen type I and collagen 1 type II staining and goat anti-rabbit IgG for TUJ1, cleaved caspase 3, TRP1, and BLBP staining, respectively; 1:100 dilution) were used as secondary antibodies. For CD31 staining, either Alexa Fluor 568 (goat anti-rat IgG, Molecular Probe, 1:100 dilution) or Cy5 (goat anti-rat IgG, 1:200 dilution) was used. Images were captured using a Nikon PCM 2000 confocal microscope. For the TUJ1 staining of cardiac NCCs in vitro, neural tubes from the midotic placode to the third somite containing NCCs were excised from E8.5 embryos and explants cultured on fibronectin-coated dishes.22 Cells were cultured overnight to obtain early migrating undifferentiated NCC or for 4 days to promote neuronal differentiation. The cultures were fixed and stained using TUJ1 (1:2000) and counterstained with DAPI.23
Results
Distribution of NCCs in the Aortic Arch System
NCCs contribute to AP septation and the remodeling of arch arteries with the process completed by late embryogenesis. We used 2 neural crest specific transgenic mice, Wnt1-Cre and P0-Cre, crossed with R26R reporter mice to compare NCC distribution in the aortic arches. To exclude the potential for long-lived -gal expression complicating the analyses and to follow labeled cells directly, a CAG-CAT-EGFP reporter mouse was also generated. The data show a strong concordance of expression resulting from recombination initiated from the 2 neural crest–specific promoters (Figure 1). At late fetal stages, neural crest–derived cells are distributed in the ascending aorta, right brachiocephalic artery, left carotid artery, ductus arteriosus, and pulmonary trunk, although pulmonary trunk distribution is somewhat faint. The descending aorta and both pulmonary arteries are devoid of staining. The overall distribution patterns are identical between Wnt1-Cre::R26R and Wnt1-Cre::EGFP, indicating that -gal expression at these stages is not attributable to long-lived enzymatic activity (Figure 1A, A', B, B') and are consistent with previously described expression.9–13 Comparison of P0-Cre::R26R with Wnt1-Cre::R26R expression patterns of -gal–expressing cells in the heart verified NCC-specific distribution (Figure 1C, C').
Persistence of NCCs in the Valvular Regions During Development and Cardiac Maturation
NCCs invade both ventral and dorsal cushions in the OFT before initiation of AP septation at around E11.5.13 However, the ultimate contribution of NCCs to the valves in late development and postnatal stages is controversial.12,13,16 When we analyzed the distribution of cardiac NCCs at late fetal stages and in the adult, we found that significant numbers of NCCs persisted in both the semilunar and AV valves (Figures 2 and 3). Distribution patterns were identical between the Wnt1-Cre::R26R and P0-Cre::R26R mice.
Following AP septation, OFT cushions undergo remodeling and form the semilunar valvular leaflets. Similar to previous reports,6,13 the 2 leaflets adjacent to the AP septum contain numerous NCCs, as demonstrated by robust -gal expression (Figure 2A and 2B), whereas proportionately fewer -gal cells appear in the distal leaflet. Staining is apparent in both the pulmonary and aortic valves from as early as E13.5 (Figure 2B). Strikingly, it remains prominent at late fetal stages (E16.5, E17.5) and persists through the neonatal and into the early adult at 8 weeks (Figure 2). To visualize the presence of neural crest–derived cells in the valvular leaflets, video microscopy of the Wnt1-Cre::EGFP-positive embryo hearts (E17.5) was used to record the valvular motion (video file in the online data supplement available at http://circres.ahajournals.org). We observed the opening/closing of EGFP-positive valve leaflets throughout the cardiac cycle, further demonstrating that the cells in the valve in late development contain a significant proportion of cells of neural crest origin.
At E12.5, a small number of -gal–expressing cells in Wnt1-Cre::R26R embryos are first detected migrating into the AV canal and AV valves, and, by E13.5, these become more numerous (Figure 3A). The labeled cells visible in the whole-mounted material in all models were found exclusively in the septal leaflet, whereas the mural leaflet was almost devoid of staining (Figure 3A and 3B, left panels, and 3C). This finding was confirmed by examining histological sections (Figure 4). Again, the pattern and distribution of the labeled cells was identical in both the Wnt1-Cre::R26R and P0-Cre::R26R hearts with the cells persisting into adulthood. It is well known that NCCs migrate into the heart via the truncus arteriosus (arterial pole). Avian studies further demonstrated a second entry point for migrating NCCs, which contribute to the innervation of posterior part of the cardiac nervous system (venous pole).4,24 We asked whether NCCs migrate into the AV valves from either 1 or both entry points. Consistent with previous studies,15 some NCCs enter the heart from the venous pole at E12.5, penetrate the heart and migrate into the AV valves (Figure 5A through 5C). Histological sections at E12.5 revealed that NCCs were seen exclusively in the dorsal part of AV valves (venous pole), whereas the cells from the arterial pole also begin to migrate toward the anterior aspect of the AV valves (Figure 5D and 5E). The location of the group of NCC cells is consistent with the hypothesis that cells seen in the AV valves by E17.5 could have originated from the arterial pole (Figure 5F).
The Wnt promoter driving Cre recombinase is expressed specifically in the dorsal neural tube in the early embryonic stage. Wnt1 expression is extinguished as the crest cell lineage migrates away from the neural tube and the promoter remains silent thereafter.8,25 We wished to confirm that Cre expression was not activated at later stages. Cre immunostaining at E9.5 showed localized expression in the dorsal neural tube and overlapped with NCCs. We were unable to detect Cre expression in either migrating NCCs at any later times or in NCCs in the AV valves (supplemental Figure IA). Additionally, using a sensitive RT-PCR–based assay for Wnt1 transcripts, we found no evidence of wnt1 activity at later developmental stages (supplemental Figure IB). The results verified that NCC labeling in the AV valves was not caused by illicit Cre expression or reactivation of wnt1.
Lineage Analysis of Cardiac NCCs in the Valves
Previous in vitro analyses demonstrated that, at the onset of migration, NCCs give rise to at least 6 differentiated phenotypes: smooth muscle cells, fibroblasts, chondrocytes, sensory neurons, autonomic neurons, and pigment cells.26,27 The significance of the presence of NCCs and their persistence in the valves at late developmental stages was evaluated by immunohistochemistry using lineage-specific antibodies. Initial experiments showed that NCCs in the valvular regions did not colocalize with fibroblasts (collagen I) or chondrocytes (collagen II) (not shown). A recent report described the presence and distribution of melanocytes in the C57BL/6J mouse heart.28 Using a TRP1 antibody,29 which marks melanocytic precursors, we explored whether the EGFP positive cells were wholly or in part neural crest–derived melanocytes. Although no TRP1-positive cells were found in OFT valves in E17.5 hearts, many but not all cells in the septal leaflets of the tricuspid (Figure 6) and mitral (not shown) valves were strongly stained. The heterogeneity of coexpression, apparent at high magnification (Figure 6, panels d'-f'), indicated that not all NCCs were committed to the melanocyte lineage. Compared with late embryonic and early postnatal stages, a large number of mature melanocytes were found in the septal leaflets of AV valves in the adult (supplemental Figure II). These data confirmed that postnatal melanocytes found in the valves were derived from NCCs and that differentiation occurred postnatally.28
NCCs in the Heart Express Neurogenic Markers
Positive staining with TUJ1, a pan-immature neural marker, was found in the septal leaflets of both OFT and AV valves as well as in the AP septum (Figure 7A) and colocalized with EGFP-positive NCCs. NCC neurogenic potential was demonstrated in vitro using NCCs isolated from the dorsal neural tube of E8.5 embryos. Neural crest explants were incubated in culture for 24 hours and the emerging, undifferentiated cells stained positive for TUJ1 (Figure 7B). After 4 days in culture, when NCCs have begun to differentiate, positive TUJ1 staining along linear axonal formation was apparent, suggesting that TUJ1 may mark a subpopulation of migrating NCCs. Furthermore, sections of the AV valves taken at 2 weeks postbirth from Wnt1-Cre::EGFP-positive animals no longer stained with TUJ1 (data not shown), indicating that the cells resident in the valves are not neural derivatives.
NCCs Contribute to the Proximal Cardiac Conduction System
NCC in the mouse heart15 are reported to be contiguous with the CCS. However, their contribution to the conduction system is unclear. Although most NCCs are eliminated via apoptosis in the chick,4 mouse NCCs are proposed to play a role in CCS maturation.15 We therefore asked whether areas of the CCS also contain NCCs. When Wnt1-Cre::EGFP mice were examined at E17.5, the proximal CCS, including the posterior internodal tract, His bundle and bundle branches (Figure 8A) all contained EGFP-positive cells. In the rabbit heart, neurofilament 160 (NF160) is recognized as a unique marker for the conduction system in early development on into the adult.30 Immunostaining with NF160 and TUJ1 colocalized with EGFP-positive cells in the conduction system (supplemental Figure III). Sections stained with antibodies against activated caspase 3 showed few if any positive cells present at E12.5, E14.5, and E17.5. The few cells that were positive were not labeled with EGFP (data not shown). To further examine the nature of these cells, a marker for immature neural crest–derived neuroglia, BLBP,21 was used. BLBP staining colocalized with some but not all EGFP-positive cells and was most prominent in the CCS at the bundle branch bifurcation (Figure 8B). Immunostaining with a second immature glial marker, GLAST,31 also colocalized with EGFP-positive cells (supplemental Figure IIIB), whereas a fully differentiated glial marker, GFAP,32 did not label any of the CCS. Although we have not resolved whether specific NCCs express these different markers, our results suggest that NCCs contributing to the maturating proximal CCS express a unique mixed-cell potential.
Discussion
This study represents the first report that directly compares fate mapping of NCCs between 2 well-characterized NCC-restricted promoters using Cre-loxP recombination strategies on a homogenous genetic background. Previous recombination studies yielded valuable data on fate mapping of NCCs in vivo,8–13,15,16 complementing in vitro analyses of NCC potential to differentiate into multiple cell types.26,27 However, the contribution of NCCs in later developmental stages to valve formation and the CCS has received less attention, and NCC persistence in the adult heart, other than their contribution to the great vessels, has not been determined. Our data show that NCCs persist in the valvular leaflets in the adult heart, are intimately associated with areas of the proximal CCS, and may retain elements of multipotency in late development.
A controversial aspect of these data is that cardiac NCCs penetrate well beyond the arterial pole of the heart. In support of this conclusion, 2 independent bitransgenic labeling systems, P0-Cre and Wnt1-Cre, clearly demonstrate NCCs in both semilunar and AV valves from midgestation onward. Crest cells enter the heart from both arterial and venous poles of the heart and proceed to the AV canal and valve primordia. This is entirely consistent with retroviral labeling studies in the chick4 and corroborates Wnt1-Cre mapping in mice.15 However, our results also differ in that the NCCs are not of ventral origin. It is possible that discrepancies reflect a species difference and/or enhanced sensitivity of NCC to retroviral infection. In mice, recombination occurs early and exclusively in the dorsal neural tube (supplemental Figure I), and we find no evidence of wnt1 activity or insertional reactivation of recombinase at later developmental stages, which might label a second population of migratory cells.
It is somewhat surprising that few of the other recombination studies,13,15,16 which have used a variety of neural crest–specific promoters including Wnt1-Cre, have described labeled cells in the AV valves. There are a number of possibilities to explain this discordance in the data. Several authors reported that staining of NCCs mediated by Wnt1-Cre recombination diminished over time and disappeared by E17.5.13,15 However, very few investigators performed these studies on a pure background, and a decrease in recombination efficiency may be associated with a mixed genetic background of either the reporter or recombinase mice. For example, background differences and genetic modifiers may be critical to Pax3-Cre–mediated recombination in a knock-in model, with the mixed strain 129/SV::C57BL/6J revealing unexpected derivatives and alterations in null mouse survival.11 The genetic background of all transgenics used in this study is identical (C57BL/6J), eliminating these potentially confounding variables. It is also possible that, based on early reports of loss of expression, later stages were not a focus and exhaustively evaluated. We have recently learned that labeled NCCs that are HtpA-Cre12 recombined are abundantly apparent in the AV valves from E12.5 onward, in agreement with our observations in this report (personal communication, S. Dufour and D. Henderson, 2006). Thus depending on genetic background, labeled NCC can contribute the AV valves using three of four distinct systems for recombination mediated labeling of NCC.
The precise role of NCCs in the CCS remains unclear. In the distal cardiac conduction system of the chick, Purkinje fibers differentiate from a subset of cardiomyocytes,33,34 although it has been hypothesized that components of the proximal CCS may originate from NCCs.35 Viral and genetic mapping studies suggest a close association between NCC and the developing conduction system,4,15 which our data confirm. In the rabbit heart, NF160 expression marks both the embryonic and adult conduction systems. Vitadello et al described coexpression of NF160 and desmin in conduction myocytes and suggested that they may originate from neuroectoderm.36 However, Schwann cells, particularly nonmyelinating cells, also express both NF160 and -tubulin.37 We see that markers for undifferentiated glia (BLBP and GLAST) appear to be coexpressed with neural markers (NF160 and TUJ1) in NCCs in the His bundle and bundle branches. Although it is possible that NCCs form the neural marker labeled conducting myocardium, it is unlikely, as this labeling pattern occurs only at late developmental stages. More likely, neurofilament labeling represents components of autonomic postganglionic neurons.38 Until recently, there have been no direct data demonstrating a role for NCC in CSS maturation. Targeted knockout of HF-1b in neural crest results in dysfunctional AV conduction, presumably attributable to defective innervation,39 whereas NCC ablation in the chick resulted in the His bundles failing to undergo compaction and electrical insulation of the CCS.40 Our data show that NCCs reside in the proximal but not distal CCS and maintain both neurogenic and gliagenic potential. We think it is likely that NCCs provide neural connections and the gliagenic cells organize compaction of the His bundles and branches.
Recent studies have demonstrated that multipotent NCCs persist in several adult tissues, including PNS ganglia,41 skin,42 and the heart.17 In the latter study, P0-Cre::EGFP- labeled stem cells were directly isolated from the adult heart.17 The undifferentiated, EGFP-labeled NCCs we see in late fetal/early neonatal hearts may contribute to the described crèche of stem cells resident in the adult heart. Our immunohistochemical analysis showed that NCCs in the late fetal heart express early, often overlapping markers for melanocytic or neurogenic and gliagenic potential. On leaving the neural epithelium, NCCs undergo a progressive restriction in developmental potential and cells with various intermediate phenotypes occur along the path toward an ultimate differentiated state.43 Although we have no direct experimental evidence that supports multipotentiality of the labeled NCCs, we speculate that these cells are not fully differentiated at this stage, as evidenced by the lack of mature markers, and may represent cells somewhere along the developmental pathway.
Acknowledgments
Sources of Funding
This work was supported by NIH grants HL69779, HL56370, HL074728, HL61638, and HL52318 (to J.R.).
Disclosures
None.
Footnotes
Original received November 30, 2005; revision received May 4, 2006; accepted May 9, 2006.
References
Kirby ML, Gale TF, Stewart DE. Neural crest cells contribute to normal aorticopulmonary septation. Science. 1983; 220: 1059–1061.
Hutson MR, Kirby ML. Neural crest and cardiovascular development: a 20-year perspective. Birth Defects Res C Embryo Today. 2003; 69: 2–13. [Order article via Infotrieve]
LeDourin NM, Kalcheim G. The Neural Crest. 2nd ed. Cambridge, UK: Cambridge University Press; 1999.
Poelmann RE, Gittenberger-de Groot AC. A subpopulation of apoptosis-prone cardiac neural crest cells targets to the venous pole: multiple functions in heart development Dev Biol. 1999; 207: 271–286. [Order article via Infotrieve]
Fukiishi Y, Morriss-Kay GM. Migration of cranial neural crest cells to the pharyngeal arches and heart in rat embryos. Cell Tissue Res. 1992; 268: 1–8. [Order article via Infotrieve]
Waldo KL, Lo CW, Kirby ML. Connexin 43 expression reflects neural crest patterns during cardiovascular development. Dev Biol. 1999; 208: 307–323. [Order article via Infotrieve]
Sauer B. Inducible gene targeting in mice using the Cre/lox system. Methods. 1998; 14: 381–392. [Order article via Infotrieve]
Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol. 1998; 8: 1323–1326. [Order article via Infotrieve]
Yamauchi Y, Abe K, Mantani A, Hitoshi Y, Suzuki M, Osuzu F, Kuratani S, Yamamura K. A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice. Dev Biol. 1999; 212: 191–203. [Order article via Infotrieve]
Li J, Chen F, Epstein JA. Neural crest expression of Cre recombinase directed by the proximal Pax3 promoter in transgenic mice. Genesis. 2000; 26: 162–164. [Order article via Infotrieve]
Engleka KA, Gitler AD, Zhang M, Zhou DD, High FA, Epstein JA. Insertion of Cre into the Pax3 locus creates a new allele of Splotch and identifies unexpected Pax3 derivatives. Dev Biol. 2005; 280: 396–406. [Order article via Infotrieve]
Pietri T, Eder O, Blanche M, Thiery JP, Dufour S. The human tissue plasminogen activator-Cre mouse: a new tool for targeting specifically neural crest cells and their derivatives in vivo. Dev Biol. 2003; 259: 176–187. [Order article via Infotrieve]
Jiang X, Rowitch DH, Soriano P, McMahon AP, Sucov HM. Fate of the mammalian cardiac neural crest. Development. 2000; 127: 1607–1616.
Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999; 21: 70–71. [Order article via Infotrieve]
Poelmann RE, Jongbloed MR, Molin DG, Fekkes ML, Wang Z, Fishman GI, Doetschman T, Azhar M, Gittenberger-de Groot AC. The neural crest is contiguous with the cardiac conduction system in the mouse embryo: a role in induction Anat Embryol (Berl). 2004; 208: 389–393. [Order article via Infotrieve]
de Lange FJ, Moorman AF, Anderson RH, Manner J, Soufan AT, de Gier-de Vries C, Schneider MD, Webb S, van den Hoff MJ, Christoffels VM. Lineage and morphogenetic analysis of the cardiac valves. Circ Res. 2004; 95: 645–654.
Tomita Y, Matsumura K, Wakamatsu Y, Matsuzaki Y, Shibuya I, Kawaguchi H, Ieda M, Kanakubo S, Shimazaki T, Ogawa S, Osumi N, Okano H, Fukuda K. Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart. J Cell Biol. 2005; 170: 1135–1146.
Araki K, Araki M, Miyazaki J, Vassalli P. Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. Proc Natl Acad Sci U S A. 1995; 92: 160–164.
Colbert MC, Kirby ML, Robbins J. Endogenous retinoic acid signaling colocalizes with advanced expression of the adult smooth muscle myosin heavy chain isoform during development of the ductus arteriosus. Circ Res. 1996; 78: 790–798.
Wang X, Osinska H, Dorn GW, 2nd, Nieman M, Lorenz JN, Gerdes AM, Witt S, Kimball T, Gulick J, Robbins J. Mouse model of desmin-related cardiomyopathy. Circulation. 2001; 103: 2402–2407.
Feng L, Hatten ME, Heintz N. Brain lipid-binding protein (BLBP): a novel signaling system in the developing mammalian CNS. Neuron. 1994; 12: 895–908. [Order article via Infotrieve]
Li J, Molkentin JD, Colbert MC. Retinoic acid inhibits cardiac neural crest migration by blocking c-Jun N-terminal kinase activation. Dev Biol. 2001; 232: 351–361. [Order article via Infotrieve]
Wang L, Mear JP, Kuan CY, Colbert MC. Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cells. Dev Growth Differ. 2005; 47: 119–130. [Order article via Infotrieve]
Verberne ME, Gittenberger-de Groot AC, Poelmann RE. Lineage and development of the parasympathetic nervous system of the embryonic chick heart. Anat Embryol (Berl). 1998; 198: 171–184. [Order article via Infotrieve]
Echelard Y, Vassileva G, McMahon AP. Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS. Development. 1994; 120: 2213–2224.
Ito K, Sieber-Blum M. Pluripotent and developmentally restricted neural-crest-derived cells in posterior visceral arches. Dev Biol. 1993; 156: 191–200. [Order article via Infotrieve]
Sieber-Blum M. Cardiac neural crest stem cells. Anat Rec A Discov Mol Cell Evol Biol. 2004; 276: 34–42. [Order article via Infotrieve]
Mjaatvedt CH, Kern CB, Norris RA, Fairey S, Cave CL. Normal distribution of melanocytes in the mouse heart. Anat Rec A Discov Mol Cell Evol Biol. 2005; 285: 748–757. [Order article via Infotrieve]
Steel KP, Davidson DR, Jackson IJ. TRP-2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development. 1992; 115: 1111–1119.
Gorza L, Vitadello M. Distribution of conduction system fibers in the developing and adult rabbit heart revealed by an antineurofilament antibody. Circ Res. 1989; 65: 360–369.
Shibata T, Yamada K, Watanabe M, Ikenaka K, Wada K, Tanaka K, Inoue Y. Glutamate transporter GLAST is expressed in the radial glia-astrocyte lineage of developing mouse spinal cord. J Neurosci. 1997; 17: 9212–9219.
Heins N, Malatesta P, Cecconi F, Nakafuku M, Tucker KL, Hack MA, Chapouton P, Barde YA, Gotz M. Glial cells generate neurons: the role of the transcription factor Pax6. Nat Neurosci. 2002; 5: 308–315. [Order article via Infotrieve]
Cheng G, Litchenberg WH, Cole GJ, Mikawa T, Thompson RP, Gourdie RG. Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage. Development. 1999; 126: 5041–5049.
Gourdie RG, Mima T, Thompson RP, Mikawa T. Terminal diversification of the myocyte lineage generates Purkinje fibers of the cardiac conduction system. Development. 1995; 121: 1423–1431.
Gorza L, Schiaffino S, Vitadello M. Heart conduction system: a neural crest derivative Brain Res. 1988; 457: 360–366. [Order article via Infotrieve]
Vitadello M, Vettore S, Lamar E, Chien KR, Gorza L. Neurofilament M mRNA is expressed in conduction system myocytes of the developing and adult rabbit heart. J Mol Cell Cardiol. 1996; 28: 1833–1844. [Order article via Infotrieve]
Roberson MD, Toews AD, Goodrum JF, Morell P. Neurofilament and tubulin mRNA expression in Schwann cells. J Neurosci Res. 1992; 33: 156–162. [Order article via Infotrieve]
Kirby ML. Cardiac Development. Oxford, UK: Oxford University Press; 2006.
St Amand TR, Lu JT, Zamora M, Gu Y, Stricker J, Hoshijima M, Epstein JA, Ross JJ Jr, Ruiz-Lozano P, Chien KR. Distinct roles of HF-1b/Sp4 in ventricular and neural crest cells lineages affect cardiac conduction system development. Dev Biol. 2006; 291: 208–217. [Order article via Infotrieve]
Gurjarpadhye A, Hewett KW, Justus C, Wen X, Kirby ML, Gourdie RG. Cardiac neural crest cells are necessary for compaction and function of developing conduction system bundles. Presented at the 12th Annual Weinstein Cardiovascular Development Conference, May 19–22, 2005, Tuscon, Ariz. S34 Abstract.
Morrison SJ, White PM, Zock C, Anderson DJ. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell. 1999; 96: 737–749. [Order article via Infotrieve]
Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F, Sadikot A, Kaplan DR, Miller FD. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol. 2001; 3: 778–784. [Order article via Infotrieve]
Le Douarin NM, Dupin E. Multipotentiality of the neural crest. Curr Opin Genet Dev. 2003; 13: 529–536. [Order article via Infotrieve]
the Department of Pediatrics, Division of Molecular Cardiovascular Biology, Children’s Hospital Research Foundation, Cincinnati, Ohio.
Abstract
Multipotent neural crest cells (NCCs) are a major extracardiac component of cardiovascular development. Although recognized as contributing cells to the arterial valves at early developmental stages, NCC persistence in the valves at later times or in the adult heart is controversial. We analyzed NCC persistence and contributions to both semilunar and atrioventricular (AV) valves in the mature heart. Two NCC-specific promoters driving Cre recombinase, Wnt1-Cre and P0-Cre, were mated with floxed reporter mice, R26R or CAG-CAT-EGFP, to map NCC fate. Hearts were analyzed before aorticopulmonary (AP) septation through adult stages. As previously demonstrated, strong NCC labeling was detected in ventral and dorsal outflow cushions before AP septation. In contrast to previous reports, we found that substantial numbers of labeled cells persisted in the semilunar valves in late fetal, neonatal, and adult hearts. Furthermore, NCCs were also found in the AV valves, almost exclusively in the septal leaflets. NCCs in the AV valves expressed melanocytic and neurogenic markers. However, cells labeled in the proximal cardiac conduction system exhibited neurogenic and gliagenic markers, whereas some NCCs expressed no differentiation specific markers. These results suggest that cardiac NCCs contribute to the mature valves and the cardiac conduction system and retain multipotent characteristics late in development.
Key Words: development heart valves embryonic development transgenic mice genetics
Introduction
The importance of the neural crest contribution to cardiovascular development has been recognized for more than 20 years.1,2 Neural crest cells (NCCs) provide a major extracardiac source of mesenchyme to the outflow tract (OFT) and postganglionic neural progenitors that innervate the heart. Seminal work using NCC extirpation and analysis of quail chick chimeras demonstrated that NCCs contributed to the aorticopulmonary (AP) septum, the tunica media of the great arteries, the OFT septum, and the semilunar valves. However, crest-derived cells were seldom found beyond the atrioventricular (AV) canal or in the AV valves and their association with the cardiac conduction system (CCS) remains controversial.
The fate of these unique migratory, multipotential cells has been studied using extrinsic and intrinsic marking methods to facilitate mapping their developmental progress.3 Most of the early fate-mapping analyses were performed using avian or amphibian chimeric embryos, with more recent studies using retroviral labeling in chick embryos.4 Fukiishi and Morriss-Kay were the first to use vital dye methods to follow cranial NCC migration in mammals.5 In recent years, promoter–reporter constructs6 or double-transgenic analysis7 using lacZ or GFP reporter mice and neural crest–specific Cre recombinase has facilitated genetic marking of neural crest in mice. Several different transgenic lines carry neural crest–specific Cre recombinase activity, including Wnt1,8 P0,9 Pax3,10,11 and HtPA.12
These model systems have provided new data on neural crest behavior in mice but have also caused some confusion. Jiang et al13 established a standard for following neural crest–derived cells during cardiovascular development, demonstrating that -gal expression in Wnt1-Cre::R26R14–positive embryos reproduced or expanded most of the patterns of neural crest–invested tissues and structures described in quail chick chimeras.6 Subsequently, other investigators used this double transgenic model to follow NCC migration into the heart15 or analyze cell lineage in cardiac valves.16 Other models of NCC-restricted recombination using P0, Pax3, or HtPA with lacZ-based reporter mice show consistent major crest contribution to the great vessels, OFT septum and semilunar valves.9–12 Most differences among the data occur in the quantitative levels of recombination or perdurance of expression. For example, the number or intensity of labeled cells in the aortic valves range from insubstantial using Wnt1-Cre recombination13 to significant cell labeling in the aortic and pulmonary valves, as reported in HtPA-Cre recombined mice.12 However qualitative differences in expression were noted in the same model of recombination. Poelmann et al15 briefly described contiguous -gal expression in the proximal conduction system and bundle branches in Wnt1-Cre::R26R embryos that was not described by Jiang et al.13 Therefore, inconsistencies in the developmental expression patterns of neural crest–derived cells exist among models, even when the same recombination system is used.
In this report, we revisit neural crest lineage tracing, using 2 well-characterized neural crest–specific promoters for recombination, Wnt1-Cre8 and P0-Cre,9 and 2 distinct reporter mice, R26R for -gal expression14 and a floxed enhanced green fluorescent protein (EGFP) reporter, to examine crest in situ. This approach facilitated direct comparison between these models at similarly staged developmental times. Furthermore, we bred both recombinase and reporter mice onto a homogeneous C57BL/6J background to eliminate any genetic variation in Cre activity, the behavior of NCCs or reporter expression. As the data are consistent among most models at early developmental stages through midgestation, we concentrated our analyses on the distribution of neural crest–derived cells during later gestation–neonatal stages and in adult hearts. In contrast to prior reports, we see NCCs in AV valves and in late stage expression in the maturing CCS. Although some neural crest–derived cells exhibit melanocytic, neurogenic, and gliagenic characteristics, not all are stained with specific markers, suggesting some may contribute to the newly described neural crest stem cell population in the adult heart.17
Materials and Methods
Transgenic Animals
Animal use was in accordance with Institutional Guidelines with all experiments reviewed and approved by the Institutional Animal Care Committee. Mice were obtained either from our preexisting colonies or, for the PO-Cre animals, From Erik Meyers, Duke University. Fate mapping used a Cre-loxP recombination strategy from embryonic day 11.5 (E11.5) through 8 weeks postnatally. Wnt1-Cre8 and Rosa26 (R26R)14 mice were bred onto the C57BL/6J background for more than 10 generations as were the other lines used in these studies. Wnt1-Cre and P0-Cre, which specifically label NCCs and their derivatives, were mated with either R26R or CAG-CAT-EGFP reporter mice. The CAG-CAT-EGFP reporter construct was generated by inserting a 3.2kb SalI–BamHI fragment containing the CMV- actin promoter and loxP flanked CAT gene18 upstream of the enhanced green fluorescent protein (EGFP) cassette. Timed pregnancies were established with the morning of a vaginal mucous plug defined as E0.5.
-Gal Expression
The Wnt1-Cre::R26R and P0-Cre::R26R mice were prepared and processed for -gal expression as described19 with the following modifications. The thoracic viscera were removed and drop fixed in 0°C 2% paraformaldehyde, 0.2% glutaraldehyde in PBS (pH 7.4). Tissues were washed extensively with PBS and immersed in a PBS/substrate solution containing 2 mmol/L MgCl2, 5 mmol/L potassium ferricyanide, 5 mmol/L ferrocyanide, and 0.1% X-gal dissolved in dimethyl formamide. For frozen sections, tissues were postfixed with 4% paraformaldehyde in PBS, infiltrated with sucrose, and embedded in OCT. Serial 8-μm sections were cut for embryos younger than E13.5 and 10-μm sections for mice older than E14.5. The samples were counterstained with nuclear fast red.
Immunohistochemistry
Wnt1-Cre::EGFP hearts were fixed overnight in 0°C 4% paraformaldehyde in PBS and prepared for immunohistochemistry as described.20 Primary antibodies used were: (1) rat anti-CD31 (1:100 dilution, BD biosciences) to detect endothelial cells; (2) goat anti–procollagen type I (1:100 dilution, Santa Cruz Biotechnology Inc) to identify fibroblasts; (3) goat anti–collagen 1 type II (1:100 dilution, Santa Cruz Biotechnology Inc) to identify chondrocytes; (4) rabbit anti–cleaved caspase 3 (1:100 dilution, Cell Signaling Technology) to detect apoptotic cells; (5) rabbit anti–tyrosinase-related protein 1 (TRP1) (1:50 dilution, Santa Cruz Biotechnology Inc) to identify melanocytes and melanocytic precursors; (6) rabbit anti–neuronal class III -tubulin (TUJ1) (1:2000 dilution, Covance) to identify neurogenic cells; (7) rabbit anti–brain lipid–binding protein (BLBP) (1:1000 dilution), which identifies radial glia.21 Alexa Fluor 568 (donkey anti-goat IgG for procollagen type I and collagen 1 type II staining and goat anti-rabbit IgG for TUJ1, cleaved caspase 3, TRP1, and BLBP staining, respectively; 1:100 dilution) were used as secondary antibodies. For CD31 staining, either Alexa Fluor 568 (goat anti-rat IgG, Molecular Probe, 1:100 dilution) or Cy5 (goat anti-rat IgG, 1:200 dilution) was used. Images were captured using a Nikon PCM 2000 confocal microscope. For the TUJ1 staining of cardiac NCCs in vitro, neural tubes from the midotic placode to the third somite containing NCCs were excised from E8.5 embryos and explants cultured on fibronectin-coated dishes.22 Cells were cultured overnight to obtain early migrating undifferentiated NCC or for 4 days to promote neuronal differentiation. The cultures were fixed and stained using TUJ1 (1:2000) and counterstained with DAPI.23
Results
Distribution of NCCs in the Aortic Arch System
NCCs contribute to AP septation and the remodeling of arch arteries with the process completed by late embryogenesis. We used 2 neural crest specific transgenic mice, Wnt1-Cre and P0-Cre, crossed with R26R reporter mice to compare NCC distribution in the aortic arches. To exclude the potential for long-lived -gal expression complicating the analyses and to follow labeled cells directly, a CAG-CAT-EGFP reporter mouse was also generated. The data show a strong concordance of expression resulting from recombination initiated from the 2 neural crest–specific promoters (Figure 1). At late fetal stages, neural crest–derived cells are distributed in the ascending aorta, right brachiocephalic artery, left carotid artery, ductus arteriosus, and pulmonary trunk, although pulmonary trunk distribution is somewhat faint. The descending aorta and both pulmonary arteries are devoid of staining. The overall distribution patterns are identical between Wnt1-Cre::R26R and Wnt1-Cre::EGFP, indicating that -gal expression at these stages is not attributable to long-lived enzymatic activity (Figure 1A, A', B, B') and are consistent with previously described expression.9–13 Comparison of P0-Cre::R26R with Wnt1-Cre::R26R expression patterns of -gal–expressing cells in the heart verified NCC-specific distribution (Figure 1C, C').
Persistence of NCCs in the Valvular Regions During Development and Cardiac Maturation
NCCs invade both ventral and dorsal cushions in the OFT before initiation of AP septation at around E11.5.13 However, the ultimate contribution of NCCs to the valves in late development and postnatal stages is controversial.12,13,16 When we analyzed the distribution of cardiac NCCs at late fetal stages and in the adult, we found that significant numbers of NCCs persisted in both the semilunar and AV valves (Figures 2 and 3). Distribution patterns were identical between the Wnt1-Cre::R26R and P0-Cre::R26R mice.
Following AP septation, OFT cushions undergo remodeling and form the semilunar valvular leaflets. Similar to previous reports,6,13 the 2 leaflets adjacent to the AP septum contain numerous NCCs, as demonstrated by robust -gal expression (Figure 2A and 2B), whereas proportionately fewer -gal cells appear in the distal leaflet. Staining is apparent in both the pulmonary and aortic valves from as early as E13.5 (Figure 2B). Strikingly, it remains prominent at late fetal stages (E16.5, E17.5) and persists through the neonatal and into the early adult at 8 weeks (Figure 2). To visualize the presence of neural crest–derived cells in the valvular leaflets, video microscopy of the Wnt1-Cre::EGFP-positive embryo hearts (E17.5) was used to record the valvular motion (video file in the online data supplement available at http://circres.ahajournals.org). We observed the opening/closing of EGFP-positive valve leaflets throughout the cardiac cycle, further demonstrating that the cells in the valve in late development contain a significant proportion of cells of neural crest origin.
At E12.5, a small number of -gal–expressing cells in Wnt1-Cre::R26R embryos are first detected migrating into the AV canal and AV valves, and, by E13.5, these become more numerous (Figure 3A). The labeled cells visible in the whole-mounted material in all models were found exclusively in the septal leaflet, whereas the mural leaflet was almost devoid of staining (Figure 3A and 3B, left panels, and 3C). This finding was confirmed by examining histological sections (Figure 4). Again, the pattern and distribution of the labeled cells was identical in both the Wnt1-Cre::R26R and P0-Cre::R26R hearts with the cells persisting into adulthood. It is well known that NCCs migrate into the heart via the truncus arteriosus (arterial pole). Avian studies further demonstrated a second entry point for migrating NCCs, which contribute to the innervation of posterior part of the cardiac nervous system (venous pole).4,24 We asked whether NCCs migrate into the AV valves from either 1 or both entry points. Consistent with previous studies,15 some NCCs enter the heart from the venous pole at E12.5, penetrate the heart and migrate into the AV valves (Figure 5A through 5C). Histological sections at E12.5 revealed that NCCs were seen exclusively in the dorsal part of AV valves (venous pole), whereas the cells from the arterial pole also begin to migrate toward the anterior aspect of the AV valves (Figure 5D and 5E). The location of the group of NCC cells is consistent with the hypothesis that cells seen in the AV valves by E17.5 could have originated from the arterial pole (Figure 5F).
The Wnt promoter driving Cre recombinase is expressed specifically in the dorsal neural tube in the early embryonic stage. Wnt1 expression is extinguished as the crest cell lineage migrates away from the neural tube and the promoter remains silent thereafter.8,25 We wished to confirm that Cre expression was not activated at later stages. Cre immunostaining at E9.5 showed localized expression in the dorsal neural tube and overlapped with NCCs. We were unable to detect Cre expression in either migrating NCCs at any later times or in NCCs in the AV valves (supplemental Figure IA). Additionally, using a sensitive RT-PCR–based assay for Wnt1 transcripts, we found no evidence of wnt1 activity at later developmental stages (supplemental Figure IB). The results verified that NCC labeling in the AV valves was not caused by illicit Cre expression or reactivation of wnt1.
Lineage Analysis of Cardiac NCCs in the Valves
Previous in vitro analyses demonstrated that, at the onset of migration, NCCs give rise to at least 6 differentiated phenotypes: smooth muscle cells, fibroblasts, chondrocytes, sensory neurons, autonomic neurons, and pigment cells.26,27 The significance of the presence of NCCs and their persistence in the valves at late developmental stages was evaluated by immunohistochemistry using lineage-specific antibodies. Initial experiments showed that NCCs in the valvular regions did not colocalize with fibroblasts (collagen I) or chondrocytes (collagen II) (not shown). A recent report described the presence and distribution of melanocytes in the C57BL/6J mouse heart.28 Using a TRP1 antibody,29 which marks melanocytic precursors, we explored whether the EGFP positive cells were wholly or in part neural crest–derived melanocytes. Although no TRP1-positive cells were found in OFT valves in E17.5 hearts, many but not all cells in the septal leaflets of the tricuspid (Figure 6) and mitral (not shown) valves were strongly stained. The heterogeneity of coexpression, apparent at high magnification (Figure 6, panels d'-f'), indicated that not all NCCs were committed to the melanocyte lineage. Compared with late embryonic and early postnatal stages, a large number of mature melanocytes were found in the septal leaflets of AV valves in the adult (supplemental Figure II). These data confirmed that postnatal melanocytes found in the valves were derived from NCCs and that differentiation occurred postnatally.28
NCCs in the Heart Express Neurogenic Markers
Positive staining with TUJ1, a pan-immature neural marker, was found in the septal leaflets of both OFT and AV valves as well as in the AP septum (Figure 7A) and colocalized with EGFP-positive NCCs. NCC neurogenic potential was demonstrated in vitro using NCCs isolated from the dorsal neural tube of E8.5 embryos. Neural crest explants were incubated in culture for 24 hours and the emerging, undifferentiated cells stained positive for TUJ1 (Figure 7B). After 4 days in culture, when NCCs have begun to differentiate, positive TUJ1 staining along linear axonal formation was apparent, suggesting that TUJ1 may mark a subpopulation of migrating NCCs. Furthermore, sections of the AV valves taken at 2 weeks postbirth from Wnt1-Cre::EGFP-positive animals no longer stained with TUJ1 (data not shown), indicating that the cells resident in the valves are not neural derivatives.
NCCs Contribute to the Proximal Cardiac Conduction System
NCC in the mouse heart15 are reported to be contiguous with the CCS. However, their contribution to the conduction system is unclear. Although most NCCs are eliminated via apoptosis in the chick,4 mouse NCCs are proposed to play a role in CCS maturation.15 We therefore asked whether areas of the CCS also contain NCCs. When Wnt1-Cre::EGFP mice were examined at E17.5, the proximal CCS, including the posterior internodal tract, His bundle and bundle branches (Figure 8A) all contained EGFP-positive cells. In the rabbit heart, neurofilament 160 (NF160) is recognized as a unique marker for the conduction system in early development on into the adult.30 Immunostaining with NF160 and TUJ1 colocalized with EGFP-positive cells in the conduction system (supplemental Figure III). Sections stained with antibodies against activated caspase 3 showed few if any positive cells present at E12.5, E14.5, and E17.5. The few cells that were positive were not labeled with EGFP (data not shown). To further examine the nature of these cells, a marker for immature neural crest–derived neuroglia, BLBP,21 was used. BLBP staining colocalized with some but not all EGFP-positive cells and was most prominent in the CCS at the bundle branch bifurcation (Figure 8B). Immunostaining with a second immature glial marker, GLAST,31 also colocalized with EGFP-positive cells (supplemental Figure IIIB), whereas a fully differentiated glial marker, GFAP,32 did not label any of the CCS. Although we have not resolved whether specific NCCs express these different markers, our results suggest that NCCs contributing to the maturating proximal CCS express a unique mixed-cell potential.
Discussion
This study represents the first report that directly compares fate mapping of NCCs between 2 well-characterized NCC-restricted promoters using Cre-loxP recombination strategies on a homogenous genetic background. Previous recombination studies yielded valuable data on fate mapping of NCCs in vivo,8–13,15,16 complementing in vitro analyses of NCC potential to differentiate into multiple cell types.26,27 However, the contribution of NCCs in later developmental stages to valve formation and the CCS has received less attention, and NCC persistence in the adult heart, other than their contribution to the great vessels, has not been determined. Our data show that NCCs persist in the valvular leaflets in the adult heart, are intimately associated with areas of the proximal CCS, and may retain elements of multipotency in late development.
A controversial aspect of these data is that cardiac NCCs penetrate well beyond the arterial pole of the heart. In support of this conclusion, 2 independent bitransgenic labeling systems, P0-Cre and Wnt1-Cre, clearly demonstrate NCCs in both semilunar and AV valves from midgestation onward. Crest cells enter the heart from both arterial and venous poles of the heart and proceed to the AV canal and valve primordia. This is entirely consistent with retroviral labeling studies in the chick4 and corroborates Wnt1-Cre mapping in mice.15 However, our results also differ in that the NCCs are not of ventral origin. It is possible that discrepancies reflect a species difference and/or enhanced sensitivity of NCC to retroviral infection. In mice, recombination occurs early and exclusively in the dorsal neural tube (supplemental Figure I), and we find no evidence of wnt1 activity or insertional reactivation of recombinase at later developmental stages, which might label a second population of migratory cells.
It is somewhat surprising that few of the other recombination studies,13,15,16 which have used a variety of neural crest–specific promoters including Wnt1-Cre, have described labeled cells in the AV valves. There are a number of possibilities to explain this discordance in the data. Several authors reported that staining of NCCs mediated by Wnt1-Cre recombination diminished over time and disappeared by E17.5.13,15 However, very few investigators performed these studies on a pure background, and a decrease in recombination efficiency may be associated with a mixed genetic background of either the reporter or recombinase mice. For example, background differences and genetic modifiers may be critical to Pax3-Cre–mediated recombination in a knock-in model, with the mixed strain 129/SV::C57BL/6J revealing unexpected derivatives and alterations in null mouse survival.11 The genetic background of all transgenics used in this study is identical (C57BL/6J), eliminating these potentially confounding variables. It is also possible that, based on early reports of loss of expression, later stages were not a focus and exhaustively evaluated. We have recently learned that labeled NCCs that are HtpA-Cre12 recombined are abundantly apparent in the AV valves from E12.5 onward, in agreement with our observations in this report (personal communication, S. Dufour and D. Henderson, 2006). Thus depending on genetic background, labeled NCC can contribute the AV valves using three of four distinct systems for recombination mediated labeling of NCC.
The precise role of NCCs in the CCS remains unclear. In the distal cardiac conduction system of the chick, Purkinje fibers differentiate from a subset of cardiomyocytes,33,34 although it has been hypothesized that components of the proximal CCS may originate from NCCs.35 Viral and genetic mapping studies suggest a close association between NCC and the developing conduction system,4,15 which our data confirm. In the rabbit heart, NF160 expression marks both the embryonic and adult conduction systems. Vitadello et al described coexpression of NF160 and desmin in conduction myocytes and suggested that they may originate from neuroectoderm.36 However, Schwann cells, particularly nonmyelinating cells, also express both NF160 and -tubulin.37 We see that markers for undifferentiated glia (BLBP and GLAST) appear to be coexpressed with neural markers (NF160 and TUJ1) in NCCs in the His bundle and bundle branches. Although it is possible that NCCs form the neural marker labeled conducting myocardium, it is unlikely, as this labeling pattern occurs only at late developmental stages. More likely, neurofilament labeling represents components of autonomic postganglionic neurons.38 Until recently, there have been no direct data demonstrating a role for NCC in CSS maturation. Targeted knockout of HF-1b in neural crest results in dysfunctional AV conduction, presumably attributable to defective innervation,39 whereas NCC ablation in the chick resulted in the His bundles failing to undergo compaction and electrical insulation of the CCS.40 Our data show that NCCs reside in the proximal but not distal CCS and maintain both neurogenic and gliagenic potential. We think it is likely that NCCs provide neural connections and the gliagenic cells organize compaction of the His bundles and branches.
Recent studies have demonstrated that multipotent NCCs persist in several adult tissues, including PNS ganglia,41 skin,42 and the heart.17 In the latter study, P0-Cre::EGFP- labeled stem cells were directly isolated from the adult heart.17 The undifferentiated, EGFP-labeled NCCs we see in late fetal/early neonatal hearts may contribute to the described crèche of stem cells resident in the adult heart. Our immunohistochemical analysis showed that NCCs in the late fetal heart express early, often overlapping markers for melanocytic or neurogenic and gliagenic potential. On leaving the neural epithelium, NCCs undergo a progressive restriction in developmental potential and cells with various intermediate phenotypes occur along the path toward an ultimate differentiated state.43 Although we have no direct experimental evidence that supports multipotentiality of the labeled NCCs, we speculate that these cells are not fully differentiated at this stage, as evidenced by the lack of mature markers, and may represent cells somewhere along the developmental pathway.
Acknowledgments
Sources of Funding
This work was supported by NIH grants HL69779, HL56370, HL074728, HL61638, and HL52318 (to J.R.).
Disclosures
None.
Footnotes
Original received November 30, 2005; revision received May 4, 2006; accepted May 9, 2006.
References
Kirby ML, Gale TF, Stewart DE. Neural crest cells contribute to normal aorticopulmonary septation. Science. 1983; 220: 1059–1061.
Hutson MR, Kirby ML. Neural crest and cardiovascular development: a 20-year perspective. Birth Defects Res C Embryo Today. 2003; 69: 2–13. [Order article via Infotrieve]
LeDourin NM, Kalcheim G. The Neural Crest. 2nd ed. Cambridge, UK: Cambridge University Press; 1999.
Poelmann RE, Gittenberger-de Groot AC. A subpopulation of apoptosis-prone cardiac neural crest cells targets to the venous pole: multiple functions in heart development Dev Biol. 1999; 207: 271–286. [Order article via Infotrieve]
Fukiishi Y, Morriss-Kay GM. Migration of cranial neural crest cells to the pharyngeal arches and heart in rat embryos. Cell Tissue Res. 1992; 268: 1–8. [Order article via Infotrieve]
Waldo KL, Lo CW, Kirby ML. Connexin 43 expression reflects neural crest patterns during cardiovascular development. Dev Biol. 1999; 208: 307–323. [Order article via Infotrieve]
Sauer B. Inducible gene targeting in mice using the Cre/lox system. Methods. 1998; 14: 381–392. [Order article via Infotrieve]
Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol. 1998; 8: 1323–1326. [Order article via Infotrieve]
Yamauchi Y, Abe K, Mantani A, Hitoshi Y, Suzuki M, Osuzu F, Kuratani S, Yamamura K. A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice. Dev Biol. 1999; 212: 191–203. [Order article via Infotrieve]
Li J, Chen F, Epstein JA. Neural crest expression of Cre recombinase directed by the proximal Pax3 promoter in transgenic mice. Genesis. 2000; 26: 162–164. [Order article via Infotrieve]
Engleka KA, Gitler AD, Zhang M, Zhou DD, High FA, Epstein JA. Insertion of Cre into the Pax3 locus creates a new allele of Splotch and identifies unexpected Pax3 derivatives. Dev Biol. 2005; 280: 396–406. [Order article via Infotrieve]
Pietri T, Eder O, Blanche M, Thiery JP, Dufour S. The human tissue plasminogen activator-Cre mouse: a new tool for targeting specifically neural crest cells and their derivatives in vivo. Dev Biol. 2003; 259: 176–187. [Order article via Infotrieve]
Jiang X, Rowitch DH, Soriano P, McMahon AP, Sucov HM. Fate of the mammalian cardiac neural crest. Development. 2000; 127: 1607–1616.
Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999; 21: 70–71. [Order article via Infotrieve]
Poelmann RE, Jongbloed MR, Molin DG, Fekkes ML, Wang Z, Fishman GI, Doetschman T, Azhar M, Gittenberger-de Groot AC. The neural crest is contiguous with the cardiac conduction system in the mouse embryo: a role in induction Anat Embryol (Berl). 2004; 208: 389–393. [Order article via Infotrieve]
de Lange FJ, Moorman AF, Anderson RH, Manner J, Soufan AT, de Gier-de Vries C, Schneider MD, Webb S, van den Hoff MJ, Christoffels VM. Lineage and morphogenetic analysis of the cardiac valves. Circ Res. 2004; 95: 645–654.
Tomita Y, Matsumura K, Wakamatsu Y, Matsuzaki Y, Shibuya I, Kawaguchi H, Ieda M, Kanakubo S, Shimazaki T, Ogawa S, Osumi N, Okano H, Fukuda K. Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart. J Cell Biol. 2005; 170: 1135–1146.
Araki K, Araki M, Miyazaki J, Vassalli P. Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. Proc Natl Acad Sci U S A. 1995; 92: 160–164.
Colbert MC, Kirby ML, Robbins J. Endogenous retinoic acid signaling colocalizes with advanced expression of the adult smooth muscle myosin heavy chain isoform during development of the ductus arteriosus. Circ Res. 1996; 78: 790–798.
Wang X, Osinska H, Dorn GW, 2nd, Nieman M, Lorenz JN, Gerdes AM, Witt S, Kimball T, Gulick J, Robbins J. Mouse model of desmin-related cardiomyopathy. Circulation. 2001; 103: 2402–2407.
Feng L, Hatten ME, Heintz N. Brain lipid-binding protein (BLBP): a novel signaling system in the developing mammalian CNS. Neuron. 1994; 12: 895–908. [Order article via Infotrieve]
Li J, Molkentin JD, Colbert MC. Retinoic acid inhibits cardiac neural crest migration by blocking c-Jun N-terminal kinase activation. Dev Biol. 2001; 232: 351–361. [Order article via Infotrieve]
Wang L, Mear JP, Kuan CY, Colbert MC. Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cells. Dev Growth Differ. 2005; 47: 119–130. [Order article via Infotrieve]
Verberne ME, Gittenberger-de Groot AC, Poelmann RE. Lineage and development of the parasympathetic nervous system of the embryonic chick heart. Anat Embryol (Berl). 1998; 198: 171–184. [Order article via Infotrieve]
Echelard Y, Vassileva G, McMahon AP. Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS. Development. 1994; 120: 2213–2224.
Ito K, Sieber-Blum M. Pluripotent and developmentally restricted neural-crest-derived cells in posterior visceral arches. Dev Biol. 1993; 156: 191–200. [Order article via Infotrieve]
Sieber-Blum M. Cardiac neural crest stem cells. Anat Rec A Discov Mol Cell Evol Biol. 2004; 276: 34–42. [Order article via Infotrieve]
Mjaatvedt CH, Kern CB, Norris RA, Fairey S, Cave CL. Normal distribution of melanocytes in the mouse heart. Anat Rec A Discov Mol Cell Evol Biol. 2005; 285: 748–757. [Order article via Infotrieve]
Steel KP, Davidson DR, Jackson IJ. TRP-2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development. 1992; 115: 1111–1119.
Gorza L, Vitadello M. Distribution of conduction system fibers in the developing and adult rabbit heart revealed by an antineurofilament antibody. Circ Res. 1989; 65: 360–369.
Shibata T, Yamada K, Watanabe M, Ikenaka K, Wada K, Tanaka K, Inoue Y. Glutamate transporter GLAST is expressed in the radial glia-astrocyte lineage of developing mouse spinal cord. J Neurosci. 1997; 17: 9212–9219.
Heins N, Malatesta P, Cecconi F, Nakafuku M, Tucker KL, Hack MA, Chapouton P, Barde YA, Gotz M. Glial cells generate neurons: the role of the transcription factor Pax6. Nat Neurosci. 2002; 5: 308–315. [Order article via Infotrieve]
Cheng G, Litchenberg WH, Cole GJ, Mikawa T, Thompson RP, Gourdie RG. Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage. Development. 1999; 126: 5041–5049.
Gourdie RG, Mima T, Thompson RP, Mikawa T. Terminal diversification of the myocyte lineage generates Purkinje fibers of the cardiac conduction system. Development. 1995; 121: 1423–1431.
Gorza L, Schiaffino S, Vitadello M. Heart conduction system: a neural crest derivative Brain Res. 1988; 457: 360–366. [Order article via Infotrieve]
Vitadello M, Vettore S, Lamar E, Chien KR, Gorza L. Neurofilament M mRNA is expressed in conduction system myocytes of the developing and adult rabbit heart. J Mol Cell Cardiol. 1996; 28: 1833–1844. [Order article via Infotrieve]
Roberson MD, Toews AD, Goodrum JF, Morell P. Neurofilament and tubulin mRNA expression in Schwann cells. J Neurosci Res. 1992; 33: 156–162. [Order article via Infotrieve]
Kirby ML. Cardiac Development. Oxford, UK: Oxford University Press; 2006.
St Amand TR, Lu JT, Zamora M, Gu Y, Stricker J, Hoshijima M, Epstein JA, Ross JJ Jr, Ruiz-Lozano P, Chien KR. Distinct roles of HF-1b/Sp4 in ventricular and neural crest cells lineages affect cardiac conduction system development. Dev Biol. 2006; 291: 208–217. [Order article via Infotrieve]
Gurjarpadhye A, Hewett KW, Justus C, Wen X, Kirby ML, Gourdie RG. Cardiac neural crest cells are necessary for compaction and function of developing conduction system bundles. Presented at the 12th Annual Weinstein Cardiovascular Development Conference, May 19–22, 2005, Tuscon, Ariz. S34 Abstract.
Morrison SJ, White PM, Zock C, Anderson DJ. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell. 1999; 96: 737–749. [Order article via Infotrieve]
Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F, Sadikot A, Kaplan DR, Miller FD. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol. 2001; 3: 778–784. [Order article via Infotrieve]
Le Douarin NM, Dupin E. Multipotentiality of the neural crest. Curr Opin Genet Dev. 2003; 13: 529–536. [Order article via Infotrieve]
您现在查看是摘要介绍页，详见ORG附件。