Endoderm contributes to endocardial composition ... - Springer Link

2 downloads 0 Views 1MB Size Report
May 20, 2014 - tribute at least partly to the formation of endocardium of cardiogenesis. Keywords Hypoblast Á Endoderm Á Endocardium Á. DiI Á Fate map Á ...
Chin. Sci. Bull. (2014) 59(22):2749–2755 DOI 10.1007/s11434-014-0366-7

csb.scichina.com www.springer.com/scp

Article

Developmental Biology

Endoderm contributes to endocardial composition during cardiogenesis Yan Li • Xiaoyu Wang • Zhenglai Ma Manli Chuai • Andrea Mu¨nsterberg • Kenneth KaHo Lee • Xuesong Yang



Received: 25 September 2013 / Accepted: 8 January 2014 / Published online: 20 May 2014 Ó Science China Press and Springer-Verlag Berlin Heidelberg 2014

Abstract Heart formation commences from a single heart tube, which fuses from bilateral primordial heart fields. The developing heart tube is composed of outer-layer myocardial cells and inner-layer endocardial cells. Several distinct populations of precardiac cells contribute to cardiac morphogenesis. However, it still remains not very clear about the lineage of endocardium at gastrulation stage. Thereby, this study focused on ascertaining the correlation between the hypoblast in gastrulation and endocardium during cardiogenesis. Firstly, the fusing heart tube morphologically is closed to endoderm-derived pharynx floor, implying the possibility that pharynx floor might be wrapped into the formation of endoderm. Secondly, HNK1 is expressed in hypoblast strongly at gastrula stage and subsequently appeared in endocardium of cardiogenesis. Moreover, fate map data displayed that DiI labeled hypoblast was also present in endocardium later on. One more evidence is chick-quail chimera of hypoblast transplantation, in which quail-hypoblast derivative could be identified in Y. Li  X. Wang  Z. Ma  X. Yang (&) Key Laboratory for Regenerative Medicine, Ministry of Education, Medical College, Jinan University, Guangzhou 510632, China e-mail: [email protected] M. Chuai Division of Cell and Developmental Biology, University of Dundee, Dundee DD1 5EH, UK A. Mu¨nsterberg School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK K. KaH. Lee Stem Cell and Regeneration Thematic Research Programme, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong, China

endocardium of cardiogenesis by QCPN antibody. In sum, our current data suggests that endoderm in gastrula contribute at least partly to the formation of endocardium of cardiogenesis. Keywords Hypoblast  Endoderm  Endocardium  DiI  Fate map  QCPN  Quail  Chick

1 Introduction As a pumping organ, the mature heart is composed of four parts: sinus venosus, atrium, ventricle and bulbus arteriosus. During early embryonic development, heart formation undergoes a series of transformation, which starts from the formation of a straight heart tube and followed by the right looping of the heart tube and septation [1]. The initial step of heart formation is to form a straight heart tube deriving from the fusion of bilateral cardiomyogenesis cells in primary/secondary heart fields on embryonic midline [2]. Although the majority of myocardial precursor cells originate from mesoderm adjacent to anterior primitive streak during early gastrulation, the continuous interaction between epiblast and hypoblast is absolutely indispensable to form an intact heart tube. It is because the crucial role of the signaling from anterior endoderm such as BMP, FGF and Wnt, which are able to induce the corresponding primitive streak cells to differentiate into cardiac myocyte during cardiomyogenesis [3, 4]. It is well known that the field of splanchnic mesoderm gives rise to the anterior & dorsal part of heart tube via the transient existence in medial cardiac crescent [5]. The outflow tract (OFT) root in the anterior part of second heart field (SHF) [6], contributes to the formation of smooth muscle to the base of the great arteries [5, 7–9]. Cardiac

123

2750

neural crest cells are one of the contributors for early heart tube formation as well. Combining with a carbohydrate epitope in cell surface glycoproteins and glycolipids, HNK-1 is expressed in migrating neural crest cells and cardiomyocytes during embryonic development [10]. Nevertheless, in this preliminary experiment of this project, we found that it was successively expressed in hypoblast at gastrula stage and cardiomyocyte including endocardium during cardiomyogenesis. Hence, HNK1 was employed for the tracking the lineage of endocardium from primitive steak stage in this study. The fate map of vital dye labeling, the traditional technique, has been used to investigate the cell fate during embryonic development since long time ago [11]. Over here, we employed it again to micro-inject DiI in hypoblast underlying primary heart field in order to track the DiI-labeled positive cells in the process of cardiogenesis. Likewise, chick-quail chimera is also a powerful approach to study the lineage of progenitor cells in early embryos [12, 13]. The mechanism behind is that quail cell derivative could be identified using QCPN (quail specific nucleolar marker) antibody in grown up embryos. In this study, all the data from morphological analysis, HNK-1 expression pattern around cardiogenesis, DiI labeling fate map and quail-chick chimera raised one possibility that hypoblast—endoderm might be able to contribute to the some component of endocardium during cardiomyogenesis.

2 Materials and methods

Chin. Sci. Bull. (2014) 59(22):2749–2755

2.3 DiI fate mapping Using pulled-glass capillaries, the hypoblast of HH3? chick embryos were labeled with DiI (Molecular Probes, 1 mg/mL in glucose) by multiple focal injection of DiI, which was performed on EC culture [15]. The site of micro-injection is in anterior hypoblast of HH3? chick embryos as schematically demonstrated in Fig. 3a. The quality of DiI labeling was confirmed by checking DiI florescence intensity and localization under a fluorescence microscope. The chick embryos with DiI-labelled hypoblast were re-incubated for 24 h prior to being fixed and cryosections, and the fate map of DiIlabelled hypoblast cells were eventually analyzed through photographing the transverse section of the developing heart tube. All DiI-labelled hypoblast experiments were performed in replicates where at least 15 embryos were used. 2.4 Chick-quail transplantation The modified grafting and staining methods in this study were employed based on previous literatures [16, 17]. Briefly, the lateral portion of hypoblast adjacent to anterior primitive streak was extirpated from a HH3?-4 chick embryo and replaced by an equivalent hypoblast from a quail embryo at exactly same HH stage [14]. The chimeras were re-incubated for 20 h more prior to fixation and serial frozen section for acetic carmine staining and immunohistochemistry. Subsequently, the quail cells derived from this hypoblast could be identified by QCPN, the antibody against quail cell nucleus specifically.

2.1 Chick and quail embryos 2.5 Immunohistochemistry on whole mount embryos The fertilized leghorn eggs were obtained from the Avian Farm of South China Agriculture University, and incubated in a humidified incubator (Yiheng Instruments, Shanghai, China) at 38 °C with humidity until the required Hamburger and Hamilton [14] stages of chick embryo. Quail fertilized eggs were raised in the animal center of Jinan University Medical School. 2.2 Acetic carmine staining Acetic carmine staining in while-mount early chick embryo was performed according to a standard protocol. Briefly, acetic carmine solution was prepared by adding 5 g carmine to 200 mL of 50 % acetic acid; boiling in water bath for 15 min prior to filtering the stain. Whole-mount chick embryos were exposed to acetic carmine stain overnight, and then washed in distilled water completely. Afterwards, the embryos were transferred to 1 % hydrochloric acid in 70 % ethanol to decolorize until the chicken morphological structure could be obviously seen in detail, and eventually the embryos were transferred to glycerin until becoming clear.

123

Immunohistochemistry against HNK1 (N-CAM, IgM) and QCPN was performed on various stages of whole mount chick embryos. Briefly, the embryos were fixed with 4 % paraformaldehyde (PFA) at 4 °C overnight, and unspecific immunoreactions was blocked with 2 % Bovine Serum Albumin (BSA) ? 1 % Triton-X ? 1 % Tween 20 in PBS for 2 h at room temperature, followed by a brief wash in PBS, the embryos were incubated with primary monoclonal antibody mixture raised against HNK1 (1:200 diluted, Sigma, USA) and QCPN (1:100 diluted, DSHB, USA) overnight at 4 °C on shaking. After extensively washing, the embryos were incubated with specific secondary antibody mixture coupled with Alexa Fluor 555 (Alexa Fluor 555 goat anti-mouse IgM and Alexa Fluor 555 goat anti-mouse IgG; 1:1000, Invitrogen, USA) overnight at 4 °C on shaking to visualize the primary antibodies. After immunohistochemistry, all the embryos were counterstained with DAPI (40 -6-Diamidino-2-phenylindole, 5 lg/mL, Invitrogen, USA) for 1 h at room temperature. Subsequently the embryos were sectioned with a cryostat microtome (Leica CM1900, Germany). The sections

Chin. Sci. Bull. (2014) 59(22):2749–2755

2751

were mounted in mounting solution (Mowiol 4-88, Sigma, USA) on glass slides and sealed with coverslips. All experiments were performed in replicates of 5–6 embryos and representative examples are presented. 2.6 Photography After immunohistochemistry whole mount embryos were photographed using stereoscope fluorescence microscope (Olympus MVX10, Germany) and imaging software (Image-Pro Plus 7.0). Sections were photographed using an epi-fluorescent microscope (Olympus IX51, Leica DM 4000B, Germany) at 2009 or 4009 magnification with the Olympus software package Leica CW4000 FISH.

3 Results 3.1 Morphological connection between pharynx floor and endocardium during heart tube formation To investigate cell sources of endocardium in the early heart tube, we carefully observed the morphogenesis of heart tube at HH9-10 chick embryos stained with acetic carmine. At Hamburger-Hamilton [14] stage HH9 chick embryo, bilateral cardiac tubes fuse to form a single straight heart tube in the ventral midline, at the same time the two sides of the foregut coalesces medially (Fig. 1a–c). This process was just evidentially shown in the transverse sections of Fig. 1a–c. In the anterior portion of heart tube, the bilateral cardiac tubes have not fully coalesced so that the midline prominence is visible (Fig. 1a), and the bilateral cardiac tubes are similarly still in a discrete state in the portion of more caudal heart tube (Fig. 1b). When the cross section of heart tube moved more caudally, the bilateral heart tube nearly fused together simultaneously, so did endocardial tube (Fig. 1c). The aforementioned phenomena could be more clearly noticed in the high magnification image (Fig. 1a0 –c0 ). It is noteworthy that the fusing cardiac tube was really closed to pharynx floor, which might imply that the some of endocardium derive from endoderm-lineage pharynx or endoderm directly. We suppose there truly is the possibility since we also can found the ligation in the older heart tube section, in which the endocardium was connected to the pharynx floor in the fused heart tube (Fig. 1d–f, d0 –f0 ). Of course, we can also not exclude the possibility that endoderm gave rise to endocardium in part directly when bilateral cardiac tube fused at midline. 3.2 The HNK1? cell expression pattern during the cardiogenesis implies the contribution of endoderm cell derivatives to endocardium To further investigate the cell lineage between endoderm and endocardium during cardiogenesis, we carefully

Fig. 1 (Color online) The localization of spatiotemporal endocardium in transverse section of developing heart tube. Acetic carmine staining for whole mount HH9-10 chick embryo performed before crosssectioning. a–c: The transverse sections of HH9 chick embryo along with anterior to posterior axis of heart tube. a0 –c0 : The magnification imagines from a–c as indicated by dotted line squares in a–c respectively, in which bilateral heart tubes are fusing and so are endocardium labeled by ec in each panel. d–f: The transverse sections of HH10 chick embryo along with anterior to posterior axis of heart tube. d0 –f0 : The magnification imagines from d–f as indicated by dotted line squares in d–f respectively, in which bilateral heart tubes have fused and so did endocardium, please note that endocardium still has close ligation with pharynx floor labeled by ec in each panels. Abbreviation: nt, neural tube; en, endoderm; ht, heart tube; ec, endocardium; ph, pharynx. Scale bar = 100 lm in a–f and 50 lm in a0 –f0

analyzed the expression pattern of HNK1 during chick embryo gastrulation. The cell component sources of heart tube formation derive from the precursor cells in three germ layer stage, the period of gastrulation of embryo development. HNK1 was chosen in this regard because it can be strongly expressed in anterior part of hypoblast, the progenitor of endoderm in gastrulation (Fig. 2a–b). Along with embryo development, the HNK1-expressed hypoblast converted to pharynx and anterior part of endoderm (Fig. 2c–d). In the following, heart tube start to form at ventral middle line, in which we supposed that the endocardium might at least partly derive from pharynx floor when bilateral heart tube fused. It might explain why HNK1 was expressed in initial endocardium despite of becoming weaker at stage of HH9-10 (Fig. 2e–h). When straight heart tube started to bend, the endocardium gradually extends and HNK1 expression became stronger than in early stage as indicated in Fig. 2i–j and then much stronger expression could be observed at C-loop heart tube

123

2752

Chin. Sci. Bull. (2014) 59(22):2749–2755

stage (Fig. 2k–l). The dynamic expression pattern of HNK1 around the heart tube formation suggests that endoderm closed related with endocardium to some extent, in another word, endoderm might partly contribute to endocardium formation during cardiogenesis. 3.3 The fate map of DiI-labeled hypoblast revealed that endoderm gave partly rise to endocardium formation

Fig. 2 (Color online) The HNK1 expression pattern transforms from endoderm at HH4 stage to endocardium at HH12 chick embryo. The whole-mount chick embryo (HH4-12) immunohistochemistry was performed against HNK1. a–b: The images of bright-field (a) and fluorescence (b) respectively at HH4 chick embryo. b0 –b00 : The transverse sections at the site of anterior and middle primitive streak as indicated by dotted lines in b. HNK1 is more strongly expressed in anterior region (b0 ) of hypoblast than the one in middle (b00 ). c–d: The images of bright-field (c) and fluorescence (d) respectively at HH8 chick embryo. d0 –d00 : The transverse sections at the head folder as indicated by dotted lines in d. HNK1 is more strongly expressed in pharynx and endoderm of head folder (d0 –d00 ). e–f: The images of bright-field (e) and fluorescence (f) respectively at HH9 chick embryo. f0 –f00 : The transverse sections at the head folder as indicated by dotted lines in f. HNK1 is especially strongly expressed in pharynx floor and endoderm of head folder (f0 –f00 ). g–h: The images of brightfield (g) and fluorescence (h) respectively at HH10 chick embryo. h0 – h00 : The transverse sections at the head folder as indicated by dotted lines in h. HNK1 is especially strongly expressed in pharynx floor and endoderm of head folder, while appearing in developing endocardium indicated by ec (h0 –h00 ). i–j: The images of bright-field (i) and fluorescence (j) respectively at HH11 chick embryo. j0 –j00 : The transverse sections at the heart tube ventricle and autumn levels as indicated by dotted lines in j. HNK1 started to express strongly in pharynx floor and splanchnic mesoderm layers, while also presented in developing endocardium indicated by ec (j0 –j00 ). k–l: The images of bright-field (k) and fluorescence (l) respectively at HH12 chick embryo. l0 –l00 : The transverse sections at the heart tube ventricle and autumn levels as indicated by dotted lines in l. HNK1 is principally expressed in endocardium indicated by ec although it could be also observed in myocardium (j0 –j00 ). DAPI staining was performed for the outline of all the transverse sections above. Abbreviation: nt, neural tube; ps, primitive streak; en, endoderm; ht, heart tube; ec, endocardium; ph, pharynx. Scale bar = 500 lm in a–l and 100 lm in b0 –b00 , d0 –d00 , f0 –f00 , j0 –j00 , l0 –l00

123

Using the fate map of traditional DiI labeling technique, we traced the DiI labeled hypoblast’s derivatives during the heart tube formation. Briefly, DiI labeling was carried out at HH4 chick embryos (Fig. 3a–b). DiI was carefully micro-injected only in hypoblast adjacent to Hensen’s node since it is the region of primary heart field in the primitive steak stage. To avoid the error from DiI labeling in wrong place, we did serially transverse-sectioning to confirm the localization of DiI-labeled cells in hypoblast only flowing the DiI micro-injection (Fig. 3c–c0 ), in which mesoderm layer was not mistakenly labeled by DiI as shown. After 20-h incubation of the DiI labeled chick embryos, the transverse section at the level of the developing heart tube demonstrated that the endocardium of the developing heart tube did contain some DiI? cells (Fig. 3e–f0 ), implying that endoderm (hypoblast in primitive streak stage) contributed at least in part to the component of endocardium formation during cardiogenesis. 3.4 The hypoblast transplantation of quail-chick also demonstrated that endoderm was involved in the formation of endocardium To further verify the lineage of endocardium with endoderm, we employed the traditional assay of quail-chick chimera since immunohistochemistry against QCPN could be utilized to identify quail-derived cell nucleus following the host chick embryo development. Since the primary heart field positioned around anterior primitive streak, a piece of quail hypoblast tissue besides anterior primitive streak was grafted to the same position of host chick embryo at the same stage as described schematically in Fig. 4a. The derivatives of quail hypoblast graft tissue were identified by QCPN immunohistochemistry following the incubation of the chimera for 20 h (Fig. 4b–f0 ). In accordance with DiI experiment, we found that QCPN? cells distributed in pharynx, endoderm (Fig. 4d0 ) and endocardium (Fig. 4e0 ). It might suggest that some of hypoblast cells gave rise to endocardium while they contribute to endoderm and pharynx. Thus, this is one more evidence that endocardium derived at least partly from hypoblast of early gastrula embryo.

Chin. Sci. Bull. (2014) 59(22):2749–2755

2753

Fig. 3 (Color online) DiI labeled hypoblast contributed partly to endocardium formation during cardiogenesis. DiI micro-injection was performed at HH4 stage chick embryo and then assessed the derivatives of DiI-labeled hypoblast (endoderm progenitor) cells in the process of cardiomyogenesis using fate map assay. a: Schematically drawing illustrated the localization of DiI labeled hypoblast in HH4 chick embryo indicated by dotted lines. b: The merge image of DiI fluorescence and bright-field of HH4 chick embryo of DiI labeling at 0 h. c–c0 : The transverse section of HH4 bright-field and DiI labeled chick embryo respectively at the site indicated by dotted line in b. In the transverse sections, endoderm position is in bottom of bright-field (c) and DiI? cells localized in hypoblast as shown in fluorescence image (c0 ). DAPI staining was performed to outline the structure. d: The schematically drawing illustrated the HH11 chick embryo 20 h incubation later after DiI labeling, in which C-loop heart tube formed. e: The merge image of DiI fluorescence and bright-field of HH11 chick embryo at the incubation of 20 h following DiI labeling. f–f0 : The transverse section of HH11 bright-field and DiI labeled chick embryo respectively at the site indicated by dotted line in e. In the transverse sections, endocardium is in top of bright-field (f) indicated by ec and DiI? cells localized in endocardium indicated by ec as shown in fluorescence image (f0 ). DAPI staining was performed to outline the structure. Abbreviation: ps, primitive streak; en, endoderm; ec, endocardium; myo, myocardium. Scale bar = 500 lm in b, e and 50 lm in c, c0 , f, f0

4 Discussion Exploring the dynamic events of cardiomyocyte differentiation involves the spatiotemporal lineage of myocardium and endocardium during early cardiogenesis. This issue is quite important because understanding it completely is conductive to master heart developmental biology mechanisms and the prospective application for heart regenerative medicine. It has been claimed that all the layers of the early heart tube at each rostra-caudal subdivision originate from the same level of primitive streak during gastrulation [18]. And subsequently the migrating precardiac heart mesoderm cells were considered to rely on the haptotaxis of fibronectin [19] or chemotaxis of FGF4&8 along the cell migration trace [20]. Thereafter, people have found that many signaling molecules such as FGF, Wnt, BMP, Shh, retinoic acid are involved in cardiogenesis [21]. Furthermore, initiating and maintaining the heart transmural patterning depend principally on endocardium rather than epicardium [22], which probably implies that there is reciprocal inductive correlation between endocardium and epicardium during the heart tube formation. As a matter of fact, there are literatures regarding the inductive interaction between the endoderm and precardiac mesoderm in

amphibians, although chick embryo are different since precardiac mesoderm cells are isolated from endoderm prior to differentiation into cardiac muscle [23]. Even though many investigations have revealed both the molecular and cellular mechanisms of cardiogenesis [21, 24, 25], the spatiotemporal development of the early heart tube wall has not been fully understood. This study focused on the cellular sources of endocardium during heart tube formation, in which heart tube is histologically composed of epimyocardium and endocardium. The developing straight heart tube consists initially of two layers, i.e., the epimyocardium in the outer layer and the endocardium in the inner layer. Obviously, both of them derive from the three germ layers during embryonic gastrula. The mesoderm cells in primary heart field originate from rostral portion of primitive streak during early gastrulation, and they are divorced from anterior primitive streak in HH3-4 chick embryo and subsequently migrates laterally and forward hinterher [26]. Using fate mapping with DiI labeling trace, Redkar et al. [26] also demonstrated that the atrium progenitor cells situated in more caudally in primitive streak in comparison to the derivation of ventricular progenitor cells in primary heart field region. After the bilateral primordial heart-forming mesoderm

123

2754

Chin. Sci. Bull. (2014) 59(22):2749–2755

Fig. 4 (Color online) Quail-derived hypoblast contributed to endocardium formation during cardiogenesis. The immunohistochemistry against QCPN was performed following the hypoblast transplantation in primary heart field of HH4 quail-chick embryos. a: The schematically drawing illustrated the localization of transplantation between quail (donor) and chick (host) embryo at HH4. Unilateral graft (hypoblast only) besides primitive streak was carried out as indicated by dotted line square. b: The schematically drawing for the embryo of quail-chick transplantation following 20 h incubation. C-loop heart tube formed at that time. c: The heart tube region for whole-mount immunohistochemistry against the QCPN in quail-chick transplantation embryo. d–d0 : The images of transverse sections of bright-field (d) and fluorescence (d0 ) respectively cross heart tube as indicated in b and c. e–e’: The magnificent images from d and d0 respectively as indicated by dotted line squares there. In the magnificent image, QCPN? cells in endocardium can be observed as indicated by ec. f–f0 : The fluorescence images of transverse sections (f) and its magnificence (f0 ) respectively cross heart tube as indicated in b and c. Here, QCPN? cells can be found in pharynx, endocardium and endoderm as indicated. Abbreviation: en, endoderm; ec, endocardium; ht, heart tube, ph, pharynx. Scale bar = 200 lm in c, 100 lm in d, d0 , f and 50 lm in e, e0 , f0

coalesce in midline, the mesoderm cell morphologically converted to cardiac contractible myocardium cells [27]. Here, the old dogma that the bilateral mesoderm heart field solely contributing to the heart myocardium needs to be accordingly revised. The emerging recognition is that only some of the contractile muscle cells in the mature heart are descendant from the primordial myocardial tissue [24]. In another word, many cell-sources contribute to the distinct myocardium layer or part during heart development. In this study, we did carefully observation for the morphological fusion process of bilateral heart tubes, and interestingly found the close correlation between pharynx floor and endocardium during the bilateral primordial heart fusion (Fig. 1), raising the possibility that part of endocardium derive from the hypoblast lineage—pharynx floor. To address the debatable hypothesis whether or not the

123

endocardial fate of endocardium is raised from mesodermal cells, which are generated from primitive streak and migrate bilaterally to the cardiac crescent, we cautiously surveyed HNK1 expression pattern from primitive streak stage to heart tube formation since it is expressed in hypoblast early and endocardium later (Fig. 2). Not only is HNK1 expressed in the both aforementioned regions, but also it strongly appears in pharynx floor. It is noteworthy that some of pharynx might be wrapped into solo primordial heart fusion to become part of endocardium (Fig. 2g– l). DiI labeling fate map is certainly conductive to address the controversial question about the origin of endocardium during early cardiogenesis. In the subsequent DiI labeling experiment, the DiI labeled hypoblast at primitive streak stage was present in endocardium when heart tube of embryo developed (Fig. 3), so that the fate map data

Chin. Sci. Bull. (2014) 59(22):2749–2755

indicated the lineage of endocardium with early endoderm—hypoblast. The conclusion could be confirmed again in the quail-chick chimera experiment, in which the hypoblast underlying primary heart field was grafted from HH3? quail to same stage chick embryo. Likewise, QCPN? cells (quail only) were also present in endocardium during heart tube formation in this kind of fate map experiment (Fig. 4e0 ). Our data again revealed previous similar finding about the projector cells of myocardium and endocardium. At least, there is possibility that both of inner and outer myocardial layers origin respectively, in which the mesodermal precursors contribute to the myocardium, while endothelial precursor cells comprise the endothelium [28–31]. There is no doubt that the more understanding the early heart tube formation, the more clearly we know congenital heart diseases, which is conductive to further cultivate new therapy or prevention for congenital heart diseases. Acknowledgements We would like to thank Dr John Chan for constructive discussions, manuscript writing and critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (30971493, 31071054), and the National Basic Research Program of China (2010CB529703). Conflict of Interest of interest.

The authors declare that they have no conflict

References 1. Linask KK (2003) Regulation of heart morphology: current molecular and cellular perspectives on the coordinated emergence of cardiac form and function. Birth Defects Res C Embryo Today 69:14–24 2. Yutzey KE, Kirby ML (2002) Wherefore heart thou? Embryonic origins of cardiogenic mesoderm. Dev Dyn 223:307–320 3. Nakajima Y, Sakabe M, Matsui H et al (2009) Heart development before beating. Anat Sci Int 84:67–76 4. Schultheiss TM, Xydas S, Lassar AB et al (1995) Induction of avian cardiac myogenesis by anterior endoderm. Development 121:4203–4214 5. Watanabe Y, Miyagawa-Tomita S, Vincent SD et al (2009) Role of mesodermal FGF8 and FGF10 overlaps in the development of the arterial pole of the heart and pharyngeal arch arteries. Circ Res 106:495–503 6. Buckingham M, Meilhac S, Zaffran S (2005) Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet 6:826–835 7. Verzi MP, McCulley DJ, De Val S et al (2005) The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field. Dev Biol 287:134–145 8. Waldo KL, Hutson MR, Stadt HA et al (2005) Cardiac neural crest is necessary for normal addition of the myocardium to the arterial pole from the secondary heart field. Dev Biol 281:66–77 9. Waldo KL, Hutson MR, Ward CC et al (2005) Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart. Dev Biol 281:78–90

2755 10. Nakajima Y, Yoshimura K, Nomura M et al (2001) Expression of HNK1 epitope by the cardiomyocytes of the early embryonic chick: in situ and in vitro studies. Anat Rec 263:326–333 11. Godement P, Vanselow J, Thanos S et al (1987) A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue. Development 101: 697–713 12. Alvarado-Mallart RM (2000) The chick/quail transplantation model to study central nervous system development. Prog Brain Res 127:67–98 13. Hallonet M, Alvarado-Mallart RM (1997) The chick/quail chimeric system: a model for early cerebellar development. Perspect Dev Neurobiol 5:17–31 14. Hamburger V, Hamilton HL (1992) A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195: 231–272 15. Streit A (2008) EC culture: a method to culture early chick embryos. Methods Mol Biol 461:255–264 16. Wilting J, Schneider M, Papoutski M et al (2000) An avian model for studies of embryonic lymphangiogenesis. Lymphology 33:81–94 17. Kurz H, Korn J, Eggli PS et al (2001) Embryonic central nervous system angiogenesis does not involve blood-borne endothelial progenitors. J Comp Neurol 436:263–274 18. Garcia-Martinez V, Schoenwolf GC (1993) Primitive-streak origin of the cardiovascular system in avian embryos. Dev Biol 159:706–719 19. Linask KK, Lash JW (1986) Precardiac cell migration: fibronectin localization at mesoderm-endoderm interface during directional movement. Dev Biol 114:87–101 20. Yang X, Dormann D, Munsterberg AE et al (2002) Cell movement patterns during gastrulation in the chick are controlled by positive and negative chemotaxis mediated by FGF4 and FGF8. Dev Cell 3:425–437 21. Rochais F, Mesbah K, Kelly RG (2009) Signaling pathways controlling second heart field development. Circ Res 104: 933–942 22. Pennisi DJ, Ballard VL, Mikawa T (2003) Epicardium is required for the full rate of myocyte proliferation and levels of expression of myocyte mitogenic factors FGF2 and its receptor, FGFR-1, but not for transmural myocardial patterning in the embryonic chick heart. Dev Dyn 228:161–172 23. Litvin J, Montgomery M, Gonzalez-Sanchez A et al (1992) Commitment and differentiation of cardiac myocytes. Trends Cardiovasc Med 2:27–32 24. Eisenberg LM, Markwald RR (2004) Cellular recruitment and the development of the myocardium. Dev Biol 274:225–232 25. Brand T (2003) Heart development: molecular insights into cardiac specification and early morphogenesis. Dev Biol 258:1–19 26. Redkar A, Montgomery M, Litvin J (2001) Fate map of early avian cardiac progenitor cells. Development 128:2269–2279 27. Colas JF, Lawson A, Schoenwolf GC (2000) Evidence that translation of smooth muscle alpha-actin mRNA is delayed in the chick promyocardium until fusion of the bilateral heart-forming regions. Dev Dyn 218:316–330 28. Abu-Issa R, Kirby ML (2007) Heart field: from mesoderm to heart tube. Annu Rev Cell Dev Biol 23:45–68 29. Baldwin HS (1996) Early embryonic vascular development. Cardiovasc Res 31:34–45 30. Brutsaert DL, De Keulenaer GW, Fransen P et al (1996) The cardiac endothelium: functional morphology, development, and physiology. Prog Cardiovasc Dis 39:239–262 31. Saga Y, Kitajima S, Miyagawa-Tomita S et al (2000) Mesp1 expression is the earliest sign of cardiovascular development. Trends Cardiovasc Med 10:345–352

123