Anatomia, Histologia, Embryologia
ORIGINAL ARTICLE
Heart Pigmentation in the Gray Bichir, Polypterus senegalus (Actinopterygii: Polypteriformes) ndez1,2 and A. C. Dura n1,2* I. Reyes-Moya1, A. Torres-Prioris1, V. Sans-Coma1,2, B. Ferna Addresses of authors:1 Department of Animal Biology, Faculty of Science, University of Malaga, 29071 Malaga, Spain; 2 Biomedical Research Institute of M alaga (IBIMA), University of Malaga, 29071 Malaga, Spain
*Correspondence: Tel.: +34 952131847; fax: +34 952131668; e-mail: [email protected] With 1 figure Received December 2013; accepted for publication October 2014 doi: 10.1111/ahe.12163
Summary The occurrence of pigment cells in the heart is well documented in amphibians, birds and mammals. By contrast, information on heart pigmentation in fish is extremely sparse. The aim is to report the presence of pigment cells over the entire surface of the heart in the gray bichir, Polypterus senegalus. The sample consisted of 12 hearts, which, after gross anatomical examination, were studied using histochemical and immunohistochemical techniques for light microscopy, and transmission electron microscopy. The pigment cells were located in the subepicardium, showing a regular distribution pattern across the whole heart, except for the anterior end of the outflow tract, where the pigmentation was much more intense. The cells contained dark, ovoid-shaped organelles which was consistent with a melanosome cell identity. As in other vertebrates, the physiological role of the pigment cells in the heart of the gray bichir is unknown. The absence of such cells in hearts of other polypteriforms suggests that cells containing melanin are not essential for normal fish heart function. Basing on literature data concerning tetrapods, it can be inferred that the pigment cells of the heart of the gray bichir derive from the neural crest. If this were true, our findings would provide the first evidence for the presence of neural crest-derived cells in the subepicardium of adult hearts of early actinopterygians.
Introduction Occurrence of pigment cells in the heart is well documented in amphibians (Moresco and Oliveira, 2009; Franco-Belussi and Oliveira, 2011; Franco-Belussi et al., 2011, 2012), birds (Brito and Kos, 2008) and mammals (Nichols and Reams, 1960; Mjaatvedt et al., 2005; Brito and Kos, 2008; Yajima and Larue, 2008; Balani et al., 2009; Levin et al., 2009; Puig et al., 2009). By contrast, information on heart pigmentation in fish is so scarce that has often been overlooked. This, together with the absence of pigmentation in the heart of fish species as emblematic in research as the zebrafish, Danio rerio, has led to the suggestion that presence of cardiac pigment cells might be associated with the four-chambered condition of the heart (Brito and Kos, 2008). However, in 1983 Benjamin et al. had already reported that melanocytes appear occasionally in the outer layer of the bulbus © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 475–480
arteriosus of the three-spined stickleback, Gasterosteus aculeatus, and ninespine stickleback, Pungitius pungitius. Later, Woodhead (1984) noticed the presence of melanomacrophages in the atrium of the guppy, Poecilia reticulata, while Leknes (1984) observed melanophores in the parietal pericardium of post-natal larvae of the same species. Santer (1985) reported that melanocytes occur occasionally in the atrial subepicardium of the furry whiptail, Trachonurus villosus. Guerrero et al. (2004) found cells containing melanin in the subepicardium of the cardiac outflow tract in alevins of the Adriatic sturgeon, Acipenser naccarii, at 8–10 days post-hatching. Dezfuli et al. (2005) observed melanocytes in the wall of capsules, formed by host’s reaction, enclosing metacercariae of Ichthyocotylurus eraticus that parasitized hearts of European white fish, Coregonus lavaretus. The aim here is to report for the first time the presence of pigment cells over the entire surface of the heart in
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fish, specifically in the gray bichir, Polypterus senegalus, a species belonging to an ancient ray-finned fish lineage, the polypteriformes, that split from the stem of the actinopterygians soon after their divergence from the sarcopterygians (Noak et al., 1996; Kikugawa et al., 2004; Takeuchi et al., 2009). Our findings were carried out in the context of a study still in progress of the heart anatomy of polypteriformes.
Materials and Methods Specimens The hearts examined belonged to 12 gray bichirs, P. senegalus, one shortfin bichir, Polypterus palmas and three redfish, Erpetoichthys calabaricus. The total length of the animals, measured from the anterior end of the head to the tip of the tail, was 7.9–10.6 cm (n = 12) in P. senegalus, 11 cm (n = 1) in P. palmas, and 26–28 cm (n = 3) in E. calabaricus. All specimens were obtained from a local supplier, and housed, separated by species, in indoor tanks of well-aerated fresh water, kept clean by means of external filter devices. Water temperature was maintained at 25°C. The animals used in this study were handled in accordance with the Spanish Regulations for the Protection of Experimental Animals (R.D. 53/2013; B.O.E. 8.2.2013). They were overanesthetized in 0.04% MS222 (tricaine methane sulphonate; Sigma Chemical Co., Poole, UK). Then, the ventral aspect of the pericardial cavity and ventral aorta were exposed by means of a longitudinal incision along the anterior, midventral line of the animal and subsequent removal of the hypobranchial musculature. The pericardial cavity was opened, and the heart was observed and photographed by means of a Leica MZ10F stereomicroscope equipped with a Leica DFC500 camera (Wetzlar, Germany). Histology For histological examination, the hearts were perfused with PBS, removed, fixed in 4% paraformaldehyde, dehydrated in graded ethanol and embedded in Histosec (Merck KGaA, Darmstadt, Germany). Serial sections of the whole heart were transversely, longitudinally or sagittally cut at 8 lm. Several unstained sections were used for direct microscopic examination to reveal pigment cells. Other sections were stained with Delafield’s haematoxylin–eosin for a general assessment of the tissue structure, with Mallory’s trichrome stain for connective tissue, or resorcin for the detection of elastin. The Schmorl’s and Lillie’s methods, as described in the work of Barka and Anderson (1963), were specifically used for the
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identification of melanin, after whitening with hydrogen peroxide. Observations were made with a Leica DMSL microscope and photographs taken using a Leica DFC500 camera. Immunohistochemistry Sections of the heart, obtained following the protocol described above, were labelled with the monoclonal antibody MF20 (Developmental Studies Hybridoma Bank, University of Iowa, USA), which recognizes an epitope in the myosin heavy chain protein. The sections were dewaxed in xylene, hydrated in an ethanol series and washed in Tris-phosphate buffered saline (TPBS, pH 7.8). Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide in TPBS for 30 min. After washing with TPBS, non-specific binding sites were saturated for 1 h with 10% sheep serum and 1% bovine serum albumin in TPBS (SB) plus 0.5% Triton X-100 (SBT). Endogenous biotin was blocked with the avidin– biotin blocking kit (Vector, Burlingame, CA, USA). Sections were washed with TPBS and then incubated overnight in the primary antibody diluted 1:50 in SBT. Control slides were incubated in SBT only. After incubation, the sections were washed in TPBS, incubated for 1.5 h at room temperature in biotin conjugated antimouse IgG (Sigma) diluted 1:100 in SBT or SB, washed again and incubated for 1 h in ExtrAvidinâ conjugate with peroxidase (Sigma) diluted 1:150 in SBT or SB. Peroxidase activity was developed with Sigma Fast 3,30 -diaminobenzidine tablets according to the instructions of the supplier. Transmission electron microscopy Heart samples containing pigment cells were fixed by immersion in 3% glutaraldehyde in PBS for 2 h at 4°C, post-fixed in 1% osmium tetroxide for 2 h at 4°C, dehydrated in graded acetone and embedded in Araldite (Fluka, Buchs, Switzerland). Ultrathin sections, obtained using an Ultracut E Reichert Jung ultratome (Wetzlar, Germany), were stained with uranyl acetate and examined with a Jeol JEM 1400 microscope (Tokyo, Japan). Results The heart of the gray bichir is composed of sinus venosus, atrium, atrioventricular region, ventricle and outflow tract arranged sequentially, through which blood flows in a caudocranial direction (Fig. 1). The outflow tract consists of two components, proximal and distal with regard to the ventricle (Fig. 1a–c). The proximal component is the conus arteriosus (Fig. 1a–c); it contains cardiac © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 475–480
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muscle in its walls and is furnished with valves at its luminal side (Fig. 1b). The distal component is a short, intrapericardial segment (Fig. 1a), devoid of cardiac muscle (Fig. 1c), which connects the conus arteriosus with the ventral aorta. All hearts of the gray bichirs included in this study were pigmented superficially, from the sinus venosus to the distal end of the outflow tract (Fig. 1a). In the cardiac segments containing myocardium, the pigment cells were distributed more or less regularly (Fig. 1a,d), even though they formed small clusters here and there. In the distal, non-myocardial portion of the outflow tract, the pigmentation was markedly more intense as a result of the massive presence of pigment cells (Fig. 1a–c). The hearts of both the shortfin bichir and redfish showed no pigmentation. As recognized by stereomicroscopy and light microscopy, the pigment cells of the gray bichir displayed an elongated, ramified or punctiform shape depending on the existence or absence of cytoplasmic processes. In all cardiac segments, the pigment cells were located in the subepicardium (Fig. 1e). They were remarkably abundant around the subepicardial coronary artery trunks running along the cardiac outflow tract (Fig. 1f,g). The pigment cells could be well identified in unstained (Fig. 1h) and stained (Fig. 1i) histological sections. After whitening the pigment cells with hydrogen peroxide, they showed a positive reactivity using both the Lillie’s (Fig. 1j) and Schmorl’s (Fig. 1k) methods, thus suggesting the presence of melanin. Transmission electron microscopy showed that the pigment cells contained dark, ovoid-shaped organelles, the appearance of which was consistent with that of melanosomes (Fig. 1l). Finally, it should be noted that the walls of the pericardial cavity and ventral aorta of the gray bichir were also remarkably pigmented (Fig. 1a). Discussion In adult mice, melanocytes may occur at different sites of the heart namely, within the mitral and tricuspid valves, including the tendinous chords, in the leaflets of the aortic valve, on the wall of the right atrium, in the atrioventricular septum and in the apex of the interventricular septum, very near or just beneath the epicardial layer (Mjaatvedt et al., 2005; Brito and Kos, 2008; Yajima and Larue, 2008; Balani et al., 2009; Levin et al., 2009). In the quail, melanin-containing cells have been found in the area of the atrioventricular valves (Brito and Kos, 2008). In several anurans, pigment cells have been reported to be present in greater or lesser amounts across the surface of the heart (Moresco and Oliveira, 2009; Franco-Belussi et al., 2011, 2012). In the 12 gray bichirs included in the © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 475–480
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present study, the pigment cells were distributed following a fairly regular pattern over the surface of all cardiac segments, from the sinus venosus to the outflow tract. Only the most distal portion of the outflow tract, devoid of myocardium, showed a notably higher concentration of pigment. In all instances, the pigment cells were located just below the epicardium. Of note is the parallel arrangement of such cells along the coronary artery trunks that crossed subepicardically the cardiac outflow tract. The pericardium and ventral aorta were also superficially pigmented. There were no pigment cells in the leaflets of the conal and atrioventricular valves. The function of the cardiac melanocytes in tetrapods is unclear. In the mouse, they may affect the viscoelastic properties of the atrioventricular valves enhancing their functioning (Balani et al., 2009). They also seem to contribute to the triggering of atrial arrhythmia (Levin et al., 2009). Moreover, anti-inflammatory activity and cytoprotection have been adduced as possible actions in mammalian hearts (reviewed in Plonka et al., 2009). Interestingly, the pigmentation of the surface of the heart and other organs increased in the anuran Eupemphix nattereri after administration of lipopolysaccharides from Escherichia coli, suggesting that the melanin performed a bactericidal function (Franco-Belussi and Oliveira, 2011). The role of the pigment cells in the heart of the gray bichir heart is an open question. Of note is that in all hearts examined, these cells were distributed following similar patterns in all individuals. This alone suggests that the pigment-containing cells must play a specific, although for the moment unknown function. On the other hand, however, the fact that there were no pigment cells in the hearts of P. palmas and E. calabaricus is rather consistent with the notion, put forward in papers concerning tetrapods (Brito and Kos, 2008; Colombo et al., 2011) that such cells are not essential for normal cardiac function. In this regard, an issue that deserves investigation concerns the possibility that cells without pigment, but belonging to the same lineage as the pigment cells in the heart of the gray bichir might appear in the subepicardium of other polypteriformes. Melanocytes occurring in mammalian hearts are supposed to originate from the neural crest. They differ morphologically from cardiomyocytes and express markers of the integumentary melanocyte lineage (reviewed in Colombo et al., 2011). The respective precursors of the cardiac melanocytes and cardiomyocytes might originate from the same axial levels and might even share migratory pathways. However, there is evidence suggesting that they constitute separate populations (Brito and Kos, 2008). To our knowledge, no evidence has been currently provided that melanin-containing cells of neural crest origin populate internal organs and/or tissues of fish. In
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Fig. 1. Gross anatomical photographs (a,f), light micrographs (b–e, g–k) and transmission electron micrograph (l) of hearts from gray bichirs. (a) Ventral view of the heart showing the distribution of the pigment cells. Note the intense pigmentation of the distal, non-myocardial component of the cardiac outflow tract (DC), and the presence of pigment cells in both the pericardium and the ventral aorta (VA). The arrowhead indicates the anterior boundary of the pericardium. A, atrium; C, conus arteriosus; PC, pericardial cavity; V, ventricle. Bar, 1 mm. (b) Longitudinal section of the anterior portion of the cardiac outflow tract stained with Mallory’s trichrome stain. The arrowheads point to the distal limit of the pericardium. Pigment cells are present along the subepicardium of both the distal, non-myocardial component (DC) of the outflow tract and the conus arteriosus (C), which contains myocardium (M) in its walls. The conal valves (asterisks) are devoid of pigment cells. Bar, 250 lm. (c) Longitudinal section of the anterior portion of the outflow tract, consecutive to that shown in panel (b), immunolabelled with MF20. Only the myocardium (M) of the conus arteriosus (C) is MF20-positive. Pigment cells are present in the subepicardium of the conus arteriosus, the distal component (DC) of the outflow tract and the ventral aorta (VA). The arrowheads indicate the anterior boundary of the pericardium. Bar, 250 lm. (d) Longitudinal section of the ventricle (V) stained with resorcin. Pigment cells occur in the whole ventricular subepicardium. Bar, 500 lm. (e) High magnification of a transverse section of the ventricle stained with resorcin. Pigment cells forming a small cluster are located in the subepicardium (SE). The myocardium (M) is devoid of pigment cells. E, epicardium. Bar, 20 lm. (f) Ventral view of the non-myocardial component (DC) of the cardiac outflow tract and the conus arteriosus (C), showing the subepicardial coronary artery trunks (CA), which are bordered by pigment cells. Bar, 250 lm. (g) High magnification of a transverse section of the conus arteriosus stained with Mallory’s trichrome stain. Two subepicardial coronary arteries (CA) can be seen, each surrounded by numerous pigment cells. M, myocardium. Bar, 20 lm. (h,i) Consecutive transverse sections of the conus arteriosus (C). Panel (h) shows an unstained histological section, where the pigment cells can be well visualized at the periphery of the conus. The inset shows a high magnification view of several clusters of pigment cells. The histological section in panel (i) is stained with Mallory’s trichrome stain. Pigment cells are present in the subepicardium, but not in the myocardium (M) or in the conal valves (asterisks). Bar, 100 lm (inset, 20 lm). (j,k) Consecutive transverse sections of the conus arteriosus (C). The section in panel (j) is stained with the Lillie’s stain and that in panel (k) with the Schmorl’s stain after whitening the pigment cells with hydrogen peroxide. In both cases, the pigment cells show positive reaction. E, epicardium; M, myocardium. Bar, 50 lm. (l) Electron micrograph of a pigment cell containing dense organelles in the cytoplasm the shape and arrangement of which are consistent with those of melanosomes. N, nucleus. Bar, 1 lm.
teleosts, non-integumentary melanin has been found in the so-called melanomacrophage centres, which are located in the stroma of hematopoietic tissues. The macrophages may obtain the melanin from phagocytosis of melanosomes formed by normally occurring melanincontaining cells (reviewed in Agius and Roberts, 2003), or by primary melanogenesis in situ (Zuasti et al., 1989, 1990), as in the spleen and liver of amphibians (Gallone et al., 2002). According to Guerrero et al. (2004), the melanocytes found in the embryonic outflow tract of the Atlantic sturgeon derived almost certainly from the neural crest. This is the sole mention to the possible morphogenetic origin of the melanin-containing cells occurring in fish hearts that we were able to find in the literature. Given this conjecture and, especially, the suggestion that the melanocytes in the mammalian and avian hearts are neural crest derivatives (Mjaatvedt et al., 2005; Brito and Kos, 2008), one is tempted to speculate that the pigment cells found in the heart of the gray bichir might also be of neural crest origin. If this were true, our observations would provide the first evidence for the presence of neural crest cells in the whole subepicardium of the adult actinopterygian heart. In addition, they would denote that the neural crest has populated extensively the whole heart from the beginning of the actinopterygian radiation. In this regard, it should be noted that in the zebrafish, the neural crest cells that migrate into the developing heart contribute to myocardial cell lineage in the atrium, ventricle and outflow tract (Li et al., 2003; Sato and Yost, 2003). We must admit, however that there is no argument to rule out that © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 475–480
the pigment cells in the heart of the bichir come from another cell lineage capable of forming melanin. A possible origin from a macrophage lineage should not be overlooked, even though there is no evidence that macrophages occur throughout the subepicardium of fish. As already discussed by Mjaatvedt et al. (2005), pluripotential cells present in the heart such as the epicardially derived cells (Wessels and Perez-Pomares, 2004), should also be taken into account, although there is no indication that they give rise to pigment cells. Acknowledgements This study was funded by the Subdirecci on General de Proyectos de Investigaci on (Ministerio de Economıa y Competitividad, Madrid, Espa~ na) and FEDER funds (contract Grant Number: CGL2010-16417). Agustina Torres Prioris is the recipient of the fellowship BES-201146901 (Ministerio de Economia y Competitividad, Espa~ na). The authors would like to thank Prof. Dr. Jose Carlos Davila, University of Malaga, for his valuable advices on transmission electron microscopy and Mr. Luis Vida, Malaga, for his technical assistance. References Agius, C., and R. J. Roberts, 2003: Melano-macrophage centres and their role in fish pathology. J. Fish Dis. 26, 499–509. Balani, K., F. C. Brito, L. Kos, and A. Agarwai, 2009: Melanocyte pigmentation stiffens murine cardiac tricuspid valve leaflet. J. R. Soc. Interface. 6, 1097–1102.
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Barka, T., and P. J. Anderson, 1963: Histochemistry: Theory, Practice and Bibliography. New York: Harper and Row. Benjamin, M., D. Norman, R. M. Santer, and D. Scarborough, 1983: Histological, histochemical, and ultrastructural studies on the bulbus arteriosus of the sticklebacks, Gasterosteus aculeatus and Pungitius pungitius (Pisces: Teleostei). J. Zool. London. 200, 325–346. Brito, F. C., and L. Kos, 2008: Timeline and distribution of melanocyte precursors in the mouse heart. Pigment Cell Melanoma Res. 21, 464–470. Colombo, S., I. Berlin, V. Delmas, and L. Larue, 2011: Classical and nonclassical melanocytes in vertebrates. In: Melanins and Melanososmes. Biosynthesis, Biogenesis, Physiological and Pathological Functions. (J. Borovansky and P. A. Riley, eds). Weinheim, Germany: Wiley-Blackwell. pp. 21–61. Dezfuli, B. S., L. Giari, E. Simoni, A. P. Shinn, M. Manera, and G. Bosi, 2005: Histopathology, ultrastructure and immunohistochemistry of Coregonus lavaretus hearts naturally infected with Ichthyocotylurus erraticus (Trematoda). Dis. Aquat. Organ. 66, 245–254. Franco-Belussi, L., and C. Oliveira, 2011: Lipopolysaccharides induce changes in the visceral pigmentation of Eupemphix nattereri (Anura: Leiuperidae). Zoology 114, 298–305. Franco-Belussi, L., L. R. S. Santos, R. Zieri, and C. Oliveira, 2011: Visceral pigmentation in four Dendropsophus species (Anura: Hylidae): occurrence and comparison. Zool. Anz. 250, 102–110. Franco-Belussi, L., L. R. S. Santos, R. Zieri, and C. Oliveira, 2012: Visceral pigmentation in three species of the genus Scinax (Anura: Hylidae): distinct morphological pattern. Anat. Rec. 295, 298–306. Gallone, A., G. Guida, I. Maida, and R. Cicero, 2002: Spleen and liver pigmented macrophages of Rana esculenta L. A new melanogenic system. Pigment Cell Res. 15, 32–40. Guerrero, A., J. M. Icardo, A. C. Duran, A. Gallego, A. Domezain, E. Colvee, and V. Sans-Coma, 2004: Differentiation of the cardiac outflow tract components in alevins of the sturgeon Acipenser naccarii (Osteichthyes, Acipenseriformes): implications for fish heart evolution. J. Morphol. 260, 172–183. Kikugawa, K., K. Katoh, S. Kuraku, H. Sakurai, O. Ishida, N. Iwabe, and T. Miyata, 2004: Basal jawed vertebrate phylogeny inferred from multiple nuclear DNA-coded genes. BMC Biol. 2, 3 (pp 1–11). Leknes, I. L., 1984: Ultrastructure of the parietal pericardium in teleosts. J. Anat. 138, 703–712. Levin, M. D., M. M. Lu, N. B. Petrenko, B. J. Hawkins, T. H. Gupta, D. Lang, P. Buckley, J. Jochems, F. Liu, C. F. Spurney, L. J. Yuan, J. T. Jacobson, C. B. Brown, L. Huang, F. Beermann, K. B. Margulies, M. Madesh, J. H. Eberwine, J. A. Epsetein, and V. V. Patel, 2009: Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers. J. Clin. Invest. 119, 3420–3436. Li, Y.-X., M. Zdanowicz, L. Young, D. Kumiski, and L. Leatherbury, 2003: Cardiac neural crest in zebrafish embryos
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contributes to myocardial cell lineage and early heart function. Dev. Dyn. 226, 540–550. Mjaatvedt, C. H., C. B. Kern, A. Norris, S. Fairey, and C. L. Cave, 2005: Normal distribution of melanocytes in the mouse heart. Anat. Rec. 285A, 748–757. Moresco, R. M., and C. Oliveira, 2009: A comparative study of the extracutaneous pigmentary system in three anuran amphibian species evaluated during the breeding season. South Am. J. Herpetol. 4, 1–8. Nichols, S. E., and W. M. Reams, 1960: The occurrence and morphogenesis of melanocytes in the connective tissues of the PET/MCV mouse strain. J. Embryol. Exp. Morphol. 8, 24–32. Noak, K., R. Zardoya, and A. Meyer, 1996: The complete mitocondrial DNA sequence of the bichir (Polypterus ornutipinnis), a basal ray-finned fish: ancient establishment of the consensus vertebrate gene order. Genetics 144, 1165– 1180. Plonka, P. M., T. Passeron, M. Brenner, D. J. Tobin, S. Shibahara, A. Thomas, A. Slominski, A. L. Kadekaro, D. Hershkovitz, E. Peters, J. J. Nordlund, Z. Abdel-Malek, K. Takeda, R. Paus, J. P. Ortonne, V. J. Hearing, and K. U. Schallreuter, 2009: What are melanocytes really doing all day long. . .? Exp. Dermatol. 18, 799–819. Puig, I., I. Yajima, J. Bonaventure, V. Delmas, and L. Larue, 2009: The tyrosinase promoter is active in a subset of vagal neural crest during early development in mice. Pigment Cell Melanoma Res. 22, 331–334. Santer, R. M., 1985: Morphology and innervation of the fish heart. Adv. Anat. Embryol. Cell Biol. 89, 1–102. Sato, M., and J. Yost, 2003: Cardiac neural crest contributes to cardiomyogenesis in zebrafish. Dev. Biol. 257, 127–139. Takeuchi, M., M. Okabe, and S. Aizawa, 2009: The genus Polypterus (Bichirs): a fish group diverged at the stem of ray-finned fishes (Actinopterygii). In: Emerging Model Organisms: A Laboratory Manual, Vol. 1. (D. A. Crotty and A. Gann, eds). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. pp. 447–467. Wessels, A., and J. M. Perez-Pomares, 2004: The epicardium and epicardially derived cells (EPDCs) as cardiac stem cells. Anat. Rec. 276A, 43–57. Woodhead, A. D., 1984: Aging changes in the heart of a poecilid fish, the guppy Poecilia reticulatus. Exp. Gerontol. 19, 383–391. Yajima, I., and L. Larue, 2008: The location of heart melanocytes is specified and the level of pigmentation in the heart may correlate with color coat. Pigment Cell Melanoma Res. 21, 471–476. Zuasti, A., J. R. Jara, C. Ferrer, and F. Solano, 1989: Occurrence of melanin granules and melanosynthesis in the kidney of Sparus auratus. Pigment Cell Res. 2, 93–99. Zuasti, A., C. Ferrer, P. Aroca, and F. Solano, 1990: Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus and Dicentrarchus labrax (Pisces, Teleostei). Pigment Cell Res. 3, 126–131.
© 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 475–480
ORIGINAL ARTICLE
Heart Pigmentation in the Gray Bichir, Polypterus senegalus (Actinopterygii: Polypteriformes) ndez1,2 and A. C. Dura n1,2* I. Reyes-Moya1, A. Torres-Prioris1, V. Sans-Coma1,2, B. Ferna Addresses of authors:1 Department of Animal Biology, Faculty of Science, University of Malaga, 29071 Malaga, Spain; 2 Biomedical Research Institute of M alaga (IBIMA), University of Malaga, 29071 Malaga, Spain
*Correspondence: Tel.: +34 952131847; fax: +34 952131668; e-mail: [email protected] With 1 figure Received December 2013; accepted for publication October 2014 doi: 10.1111/ahe.12163
Summary The occurrence of pigment cells in the heart is well documented in amphibians, birds and mammals. By contrast, information on heart pigmentation in fish is extremely sparse. The aim is to report the presence of pigment cells over the entire surface of the heart in the gray bichir, Polypterus senegalus. The sample consisted of 12 hearts, which, after gross anatomical examination, were studied using histochemical and immunohistochemical techniques for light microscopy, and transmission electron microscopy. The pigment cells were located in the subepicardium, showing a regular distribution pattern across the whole heart, except for the anterior end of the outflow tract, where the pigmentation was much more intense. The cells contained dark, ovoid-shaped organelles which was consistent with a melanosome cell identity. As in other vertebrates, the physiological role of the pigment cells in the heart of the gray bichir is unknown. The absence of such cells in hearts of other polypteriforms suggests that cells containing melanin are not essential for normal fish heart function. Basing on literature data concerning tetrapods, it can be inferred that the pigment cells of the heart of the gray bichir derive from the neural crest. If this were true, our findings would provide the first evidence for the presence of neural crest-derived cells in the subepicardium of adult hearts of early actinopterygians.
Introduction Occurrence of pigment cells in the heart is well documented in amphibians (Moresco and Oliveira, 2009; Franco-Belussi and Oliveira, 2011; Franco-Belussi et al., 2011, 2012), birds (Brito and Kos, 2008) and mammals (Nichols and Reams, 1960; Mjaatvedt et al., 2005; Brito and Kos, 2008; Yajima and Larue, 2008; Balani et al., 2009; Levin et al., 2009; Puig et al., 2009). By contrast, information on heart pigmentation in fish is so scarce that has often been overlooked. This, together with the absence of pigmentation in the heart of fish species as emblematic in research as the zebrafish, Danio rerio, has led to the suggestion that presence of cardiac pigment cells might be associated with the four-chambered condition of the heart (Brito and Kos, 2008). However, in 1983 Benjamin et al. had already reported that melanocytes appear occasionally in the outer layer of the bulbus © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 475–480
arteriosus of the three-spined stickleback, Gasterosteus aculeatus, and ninespine stickleback, Pungitius pungitius. Later, Woodhead (1984) noticed the presence of melanomacrophages in the atrium of the guppy, Poecilia reticulata, while Leknes (1984) observed melanophores in the parietal pericardium of post-natal larvae of the same species. Santer (1985) reported that melanocytes occur occasionally in the atrial subepicardium of the furry whiptail, Trachonurus villosus. Guerrero et al. (2004) found cells containing melanin in the subepicardium of the cardiac outflow tract in alevins of the Adriatic sturgeon, Acipenser naccarii, at 8–10 days post-hatching. Dezfuli et al. (2005) observed melanocytes in the wall of capsules, formed by host’s reaction, enclosing metacercariae of Ichthyocotylurus eraticus that parasitized hearts of European white fish, Coregonus lavaretus. The aim here is to report for the first time the presence of pigment cells over the entire surface of the heart in
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fish, specifically in the gray bichir, Polypterus senegalus, a species belonging to an ancient ray-finned fish lineage, the polypteriformes, that split from the stem of the actinopterygians soon after their divergence from the sarcopterygians (Noak et al., 1996; Kikugawa et al., 2004; Takeuchi et al., 2009). Our findings were carried out in the context of a study still in progress of the heart anatomy of polypteriformes.
Materials and Methods Specimens The hearts examined belonged to 12 gray bichirs, P. senegalus, one shortfin bichir, Polypterus palmas and three redfish, Erpetoichthys calabaricus. The total length of the animals, measured from the anterior end of the head to the tip of the tail, was 7.9–10.6 cm (n = 12) in P. senegalus, 11 cm (n = 1) in P. palmas, and 26–28 cm (n = 3) in E. calabaricus. All specimens were obtained from a local supplier, and housed, separated by species, in indoor tanks of well-aerated fresh water, kept clean by means of external filter devices. Water temperature was maintained at 25°C. The animals used in this study were handled in accordance with the Spanish Regulations for the Protection of Experimental Animals (R.D. 53/2013; B.O.E. 8.2.2013). They were overanesthetized in 0.04% MS222 (tricaine methane sulphonate; Sigma Chemical Co., Poole, UK). Then, the ventral aspect of the pericardial cavity and ventral aorta were exposed by means of a longitudinal incision along the anterior, midventral line of the animal and subsequent removal of the hypobranchial musculature. The pericardial cavity was opened, and the heart was observed and photographed by means of a Leica MZ10F stereomicroscope equipped with a Leica DFC500 camera (Wetzlar, Germany). Histology For histological examination, the hearts were perfused with PBS, removed, fixed in 4% paraformaldehyde, dehydrated in graded ethanol and embedded in Histosec (Merck KGaA, Darmstadt, Germany). Serial sections of the whole heart were transversely, longitudinally or sagittally cut at 8 lm. Several unstained sections were used for direct microscopic examination to reveal pigment cells. Other sections were stained with Delafield’s haematoxylin–eosin for a general assessment of the tissue structure, with Mallory’s trichrome stain for connective tissue, or resorcin for the detection of elastin. The Schmorl’s and Lillie’s methods, as described in the work of Barka and Anderson (1963), were specifically used for the
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identification of melanin, after whitening with hydrogen peroxide. Observations were made with a Leica DMSL microscope and photographs taken using a Leica DFC500 camera. Immunohistochemistry Sections of the heart, obtained following the protocol described above, were labelled with the monoclonal antibody MF20 (Developmental Studies Hybridoma Bank, University of Iowa, USA), which recognizes an epitope in the myosin heavy chain protein. The sections were dewaxed in xylene, hydrated in an ethanol series and washed in Tris-phosphate buffered saline (TPBS, pH 7.8). Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide in TPBS for 30 min. After washing with TPBS, non-specific binding sites were saturated for 1 h with 10% sheep serum and 1% bovine serum albumin in TPBS (SB) plus 0.5% Triton X-100 (SBT). Endogenous biotin was blocked with the avidin– biotin blocking kit (Vector, Burlingame, CA, USA). Sections were washed with TPBS and then incubated overnight in the primary antibody diluted 1:50 in SBT. Control slides were incubated in SBT only. After incubation, the sections were washed in TPBS, incubated for 1.5 h at room temperature in biotin conjugated antimouse IgG (Sigma) diluted 1:100 in SBT or SB, washed again and incubated for 1 h in ExtrAvidinâ conjugate with peroxidase (Sigma) diluted 1:150 in SBT or SB. Peroxidase activity was developed with Sigma Fast 3,30 -diaminobenzidine tablets according to the instructions of the supplier. Transmission electron microscopy Heart samples containing pigment cells were fixed by immersion in 3% glutaraldehyde in PBS for 2 h at 4°C, post-fixed in 1% osmium tetroxide for 2 h at 4°C, dehydrated in graded acetone and embedded in Araldite (Fluka, Buchs, Switzerland). Ultrathin sections, obtained using an Ultracut E Reichert Jung ultratome (Wetzlar, Germany), were stained with uranyl acetate and examined with a Jeol JEM 1400 microscope (Tokyo, Japan). Results The heart of the gray bichir is composed of sinus venosus, atrium, atrioventricular region, ventricle and outflow tract arranged sequentially, through which blood flows in a caudocranial direction (Fig. 1). The outflow tract consists of two components, proximal and distal with regard to the ventricle (Fig. 1a–c). The proximal component is the conus arteriosus (Fig. 1a–c); it contains cardiac © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 475–480
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muscle in its walls and is furnished with valves at its luminal side (Fig. 1b). The distal component is a short, intrapericardial segment (Fig. 1a), devoid of cardiac muscle (Fig. 1c), which connects the conus arteriosus with the ventral aorta. All hearts of the gray bichirs included in this study were pigmented superficially, from the sinus venosus to the distal end of the outflow tract (Fig. 1a). In the cardiac segments containing myocardium, the pigment cells were distributed more or less regularly (Fig. 1a,d), even though they formed small clusters here and there. In the distal, non-myocardial portion of the outflow tract, the pigmentation was markedly more intense as a result of the massive presence of pigment cells (Fig. 1a–c). The hearts of both the shortfin bichir and redfish showed no pigmentation. As recognized by stereomicroscopy and light microscopy, the pigment cells of the gray bichir displayed an elongated, ramified or punctiform shape depending on the existence or absence of cytoplasmic processes. In all cardiac segments, the pigment cells were located in the subepicardium (Fig. 1e). They were remarkably abundant around the subepicardial coronary artery trunks running along the cardiac outflow tract (Fig. 1f,g). The pigment cells could be well identified in unstained (Fig. 1h) and stained (Fig. 1i) histological sections. After whitening the pigment cells with hydrogen peroxide, they showed a positive reactivity using both the Lillie’s (Fig. 1j) and Schmorl’s (Fig. 1k) methods, thus suggesting the presence of melanin. Transmission electron microscopy showed that the pigment cells contained dark, ovoid-shaped organelles, the appearance of which was consistent with that of melanosomes (Fig. 1l). Finally, it should be noted that the walls of the pericardial cavity and ventral aorta of the gray bichir were also remarkably pigmented (Fig. 1a). Discussion In adult mice, melanocytes may occur at different sites of the heart namely, within the mitral and tricuspid valves, including the tendinous chords, in the leaflets of the aortic valve, on the wall of the right atrium, in the atrioventricular septum and in the apex of the interventricular septum, very near or just beneath the epicardial layer (Mjaatvedt et al., 2005; Brito and Kos, 2008; Yajima and Larue, 2008; Balani et al., 2009; Levin et al., 2009). In the quail, melanin-containing cells have been found in the area of the atrioventricular valves (Brito and Kos, 2008). In several anurans, pigment cells have been reported to be present in greater or lesser amounts across the surface of the heart (Moresco and Oliveira, 2009; Franco-Belussi et al., 2011, 2012). In the 12 gray bichirs included in the © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 475–480
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present study, the pigment cells were distributed following a fairly regular pattern over the surface of all cardiac segments, from the sinus venosus to the outflow tract. Only the most distal portion of the outflow tract, devoid of myocardium, showed a notably higher concentration of pigment. In all instances, the pigment cells were located just below the epicardium. Of note is the parallel arrangement of such cells along the coronary artery trunks that crossed subepicardically the cardiac outflow tract. The pericardium and ventral aorta were also superficially pigmented. There were no pigment cells in the leaflets of the conal and atrioventricular valves. The function of the cardiac melanocytes in tetrapods is unclear. In the mouse, they may affect the viscoelastic properties of the atrioventricular valves enhancing their functioning (Balani et al., 2009). They also seem to contribute to the triggering of atrial arrhythmia (Levin et al., 2009). Moreover, anti-inflammatory activity and cytoprotection have been adduced as possible actions in mammalian hearts (reviewed in Plonka et al., 2009). Interestingly, the pigmentation of the surface of the heart and other organs increased in the anuran Eupemphix nattereri after administration of lipopolysaccharides from Escherichia coli, suggesting that the melanin performed a bactericidal function (Franco-Belussi and Oliveira, 2011). The role of the pigment cells in the heart of the gray bichir heart is an open question. Of note is that in all hearts examined, these cells were distributed following similar patterns in all individuals. This alone suggests that the pigment-containing cells must play a specific, although for the moment unknown function. On the other hand, however, the fact that there were no pigment cells in the hearts of P. palmas and E. calabaricus is rather consistent with the notion, put forward in papers concerning tetrapods (Brito and Kos, 2008; Colombo et al., 2011) that such cells are not essential for normal cardiac function. In this regard, an issue that deserves investigation concerns the possibility that cells without pigment, but belonging to the same lineage as the pigment cells in the heart of the gray bichir might appear in the subepicardium of other polypteriformes. Melanocytes occurring in mammalian hearts are supposed to originate from the neural crest. They differ morphologically from cardiomyocytes and express markers of the integumentary melanocyte lineage (reviewed in Colombo et al., 2011). The respective precursors of the cardiac melanocytes and cardiomyocytes might originate from the same axial levels and might even share migratory pathways. However, there is evidence suggesting that they constitute separate populations (Brito and Kos, 2008). To our knowledge, no evidence has been currently provided that melanin-containing cells of neural crest origin populate internal organs and/or tissues of fish. In
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Fig. 1. Gross anatomical photographs (a,f), light micrographs (b–e, g–k) and transmission electron micrograph (l) of hearts from gray bichirs. (a) Ventral view of the heart showing the distribution of the pigment cells. Note the intense pigmentation of the distal, non-myocardial component of the cardiac outflow tract (DC), and the presence of pigment cells in both the pericardium and the ventral aorta (VA). The arrowhead indicates the anterior boundary of the pericardium. A, atrium; C, conus arteriosus; PC, pericardial cavity; V, ventricle. Bar, 1 mm. (b) Longitudinal section of the anterior portion of the cardiac outflow tract stained with Mallory’s trichrome stain. The arrowheads point to the distal limit of the pericardium. Pigment cells are present along the subepicardium of both the distal, non-myocardial component (DC) of the outflow tract and the conus arteriosus (C), which contains myocardium (M) in its walls. The conal valves (asterisks) are devoid of pigment cells. Bar, 250 lm. (c) Longitudinal section of the anterior portion of the outflow tract, consecutive to that shown in panel (b), immunolabelled with MF20. Only the myocardium (M) of the conus arteriosus (C) is MF20-positive. Pigment cells are present in the subepicardium of the conus arteriosus, the distal component (DC) of the outflow tract and the ventral aorta (VA). The arrowheads indicate the anterior boundary of the pericardium. Bar, 250 lm. (d) Longitudinal section of the ventricle (V) stained with resorcin. Pigment cells occur in the whole ventricular subepicardium. Bar, 500 lm. (e) High magnification of a transverse section of the ventricle stained with resorcin. Pigment cells forming a small cluster are located in the subepicardium (SE). The myocardium (M) is devoid of pigment cells. E, epicardium. Bar, 20 lm. (f) Ventral view of the non-myocardial component (DC) of the cardiac outflow tract and the conus arteriosus (C), showing the subepicardial coronary artery trunks (CA), which are bordered by pigment cells. Bar, 250 lm. (g) High magnification of a transverse section of the conus arteriosus stained with Mallory’s trichrome stain. Two subepicardial coronary arteries (CA) can be seen, each surrounded by numerous pigment cells. M, myocardium. Bar, 20 lm. (h,i) Consecutive transverse sections of the conus arteriosus (C). Panel (h) shows an unstained histological section, where the pigment cells can be well visualized at the periphery of the conus. The inset shows a high magnification view of several clusters of pigment cells. The histological section in panel (i) is stained with Mallory’s trichrome stain. Pigment cells are present in the subepicardium, but not in the myocardium (M) or in the conal valves (asterisks). Bar, 100 lm (inset, 20 lm). (j,k) Consecutive transverse sections of the conus arteriosus (C). The section in panel (j) is stained with the Lillie’s stain and that in panel (k) with the Schmorl’s stain after whitening the pigment cells with hydrogen peroxide. In both cases, the pigment cells show positive reaction. E, epicardium; M, myocardium. Bar, 50 lm. (l) Electron micrograph of a pigment cell containing dense organelles in the cytoplasm the shape and arrangement of which are consistent with those of melanosomes. N, nucleus. Bar, 1 lm.
teleosts, non-integumentary melanin has been found in the so-called melanomacrophage centres, which are located in the stroma of hematopoietic tissues. The macrophages may obtain the melanin from phagocytosis of melanosomes formed by normally occurring melanincontaining cells (reviewed in Agius and Roberts, 2003), or by primary melanogenesis in situ (Zuasti et al., 1989, 1990), as in the spleen and liver of amphibians (Gallone et al., 2002). According to Guerrero et al. (2004), the melanocytes found in the embryonic outflow tract of the Atlantic sturgeon derived almost certainly from the neural crest. This is the sole mention to the possible morphogenetic origin of the melanin-containing cells occurring in fish hearts that we were able to find in the literature. Given this conjecture and, especially, the suggestion that the melanocytes in the mammalian and avian hearts are neural crest derivatives (Mjaatvedt et al., 2005; Brito and Kos, 2008), one is tempted to speculate that the pigment cells found in the heart of the gray bichir might also be of neural crest origin. If this were true, our observations would provide the first evidence for the presence of neural crest cells in the whole subepicardium of the adult actinopterygian heart. In addition, they would denote that the neural crest has populated extensively the whole heart from the beginning of the actinopterygian radiation. In this regard, it should be noted that in the zebrafish, the neural crest cells that migrate into the developing heart contribute to myocardial cell lineage in the atrium, ventricle and outflow tract (Li et al., 2003; Sato and Yost, 2003). We must admit, however that there is no argument to rule out that © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 475–480
the pigment cells in the heart of the bichir come from another cell lineage capable of forming melanin. A possible origin from a macrophage lineage should not be overlooked, even though there is no evidence that macrophages occur throughout the subepicardium of fish. As already discussed by Mjaatvedt et al. (2005), pluripotential cells present in the heart such as the epicardially derived cells (Wessels and Perez-Pomares, 2004), should also be taken into account, although there is no indication that they give rise to pigment cells. Acknowledgements This study was funded by the Subdirecci on General de Proyectos de Investigaci on (Ministerio de Economıa y Competitividad, Madrid, Espa~ na) and FEDER funds (contract Grant Number: CGL2010-16417). Agustina Torres Prioris is the recipient of the fellowship BES-201146901 (Ministerio de Economia y Competitividad, Espa~ na). The authors would like to thank Prof. Dr. Jose Carlos Davila, University of Malaga, for his valuable advices on transmission electron microscopy and Mr. Luis Vida, Malaga, for his technical assistance. References Agius, C., and R. J. Roberts, 2003: Melano-macrophage centres and their role in fish pathology. J. Fish Dis. 26, 499–509. Balani, K., F. C. Brito, L. Kos, and A. Agarwai, 2009: Melanocyte pigmentation stiffens murine cardiac tricuspid valve leaflet. J. R. Soc. Interface. 6, 1097–1102.
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Barka, T., and P. J. Anderson, 1963: Histochemistry: Theory, Practice and Bibliography. New York: Harper and Row. Benjamin, M., D. Norman, R. M. Santer, and D. Scarborough, 1983: Histological, histochemical, and ultrastructural studies on the bulbus arteriosus of the sticklebacks, Gasterosteus aculeatus and Pungitius pungitius (Pisces: Teleostei). J. Zool. London. 200, 325–346. Brito, F. C., and L. Kos, 2008: Timeline and distribution of melanocyte precursors in the mouse heart. Pigment Cell Melanoma Res. 21, 464–470. Colombo, S., I. Berlin, V. Delmas, and L. Larue, 2011: Classical and nonclassical melanocytes in vertebrates. In: Melanins and Melanososmes. Biosynthesis, Biogenesis, Physiological and Pathological Functions. (J. Borovansky and P. A. Riley, eds). Weinheim, Germany: Wiley-Blackwell. pp. 21–61. Dezfuli, B. S., L. Giari, E. Simoni, A. P. Shinn, M. Manera, and G. Bosi, 2005: Histopathology, ultrastructure and immunohistochemistry of Coregonus lavaretus hearts naturally infected with Ichthyocotylurus erraticus (Trematoda). Dis. Aquat. Organ. 66, 245–254. Franco-Belussi, L., and C. Oliveira, 2011: Lipopolysaccharides induce changes in the visceral pigmentation of Eupemphix nattereri (Anura: Leiuperidae). Zoology 114, 298–305. Franco-Belussi, L., L. R. S. Santos, R. Zieri, and C. Oliveira, 2011: Visceral pigmentation in four Dendropsophus species (Anura: Hylidae): occurrence and comparison. Zool. Anz. 250, 102–110. Franco-Belussi, L., L. R. S. Santos, R. Zieri, and C. Oliveira, 2012: Visceral pigmentation in three species of the genus Scinax (Anura: Hylidae): distinct morphological pattern. Anat. Rec. 295, 298–306. Gallone, A., G. Guida, I. Maida, and R. Cicero, 2002: Spleen and liver pigmented macrophages of Rana esculenta L. A new melanogenic system. Pigment Cell Res. 15, 32–40. Guerrero, A., J. M. Icardo, A. C. Duran, A. Gallego, A. Domezain, E. Colvee, and V. Sans-Coma, 2004: Differentiation of the cardiac outflow tract components in alevins of the sturgeon Acipenser naccarii (Osteichthyes, Acipenseriformes): implications for fish heart evolution. J. Morphol. 260, 172–183. Kikugawa, K., K. Katoh, S. Kuraku, H. Sakurai, O. Ishida, N. Iwabe, and T. Miyata, 2004: Basal jawed vertebrate phylogeny inferred from multiple nuclear DNA-coded genes. BMC Biol. 2, 3 (pp 1–11). Leknes, I. L., 1984: Ultrastructure of the parietal pericardium in teleosts. J. Anat. 138, 703–712. Levin, M. D., M. M. Lu, N. B. Petrenko, B. J. Hawkins, T. H. Gupta, D. Lang, P. Buckley, J. Jochems, F. Liu, C. F. Spurney, L. J. Yuan, J. T. Jacobson, C. B. Brown, L. Huang, F. Beermann, K. B. Margulies, M. Madesh, J. H. Eberwine, J. A. Epsetein, and V. V. Patel, 2009: Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers. J. Clin. Invest. 119, 3420–3436. Li, Y.-X., M. Zdanowicz, L. Young, D. Kumiski, and L. Leatherbury, 2003: Cardiac neural crest in zebrafish embryos
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contributes to myocardial cell lineage and early heart function. Dev. Dyn. 226, 540–550. Mjaatvedt, C. H., C. B. Kern, A. Norris, S. Fairey, and C. L. Cave, 2005: Normal distribution of melanocytes in the mouse heart. Anat. Rec. 285A, 748–757. Moresco, R. M., and C. Oliveira, 2009: A comparative study of the extracutaneous pigmentary system in three anuran amphibian species evaluated during the breeding season. South Am. J. Herpetol. 4, 1–8. Nichols, S. E., and W. M. Reams, 1960: The occurrence and morphogenesis of melanocytes in the connective tissues of the PET/MCV mouse strain. J. Embryol. Exp. Morphol. 8, 24–32. Noak, K., R. Zardoya, and A. Meyer, 1996: The complete mitocondrial DNA sequence of the bichir (Polypterus ornutipinnis), a basal ray-finned fish: ancient establishment of the consensus vertebrate gene order. Genetics 144, 1165– 1180. Plonka, P. M., T. Passeron, M. Brenner, D. J. Tobin, S. Shibahara, A. Thomas, A. Slominski, A. L. Kadekaro, D. Hershkovitz, E. Peters, J. J. Nordlund, Z. Abdel-Malek, K. Takeda, R. Paus, J. P. Ortonne, V. J. Hearing, and K. U. Schallreuter, 2009: What are melanocytes really doing all day long. . .? Exp. Dermatol. 18, 799–819. Puig, I., I. Yajima, J. Bonaventure, V. Delmas, and L. Larue, 2009: The tyrosinase promoter is active in a subset of vagal neural crest during early development in mice. Pigment Cell Melanoma Res. 22, 331–334. Santer, R. M., 1985: Morphology and innervation of the fish heart. Adv. Anat. Embryol. Cell Biol. 89, 1–102. Sato, M., and J. Yost, 2003: Cardiac neural crest contributes to cardiomyogenesis in zebrafish. Dev. Biol. 257, 127–139. Takeuchi, M., M. Okabe, and S. Aizawa, 2009: The genus Polypterus (Bichirs): a fish group diverged at the stem of ray-finned fishes (Actinopterygii). In: Emerging Model Organisms: A Laboratory Manual, Vol. 1. (D. A. Crotty and A. Gann, eds). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. pp. 447–467. Wessels, A., and J. M. Perez-Pomares, 2004: The epicardium and epicardially derived cells (EPDCs) as cardiac stem cells. Anat. Rec. 276A, 43–57. Woodhead, A. D., 1984: Aging changes in the heart of a poecilid fish, the guppy Poecilia reticulatus. Exp. Gerontol. 19, 383–391. Yajima, I., and L. Larue, 2008: The location of heart melanocytes is specified and the level of pigmentation in the heart may correlate with color coat. Pigment Cell Melanoma Res. 21, 471–476. Zuasti, A., J. R. Jara, C. Ferrer, and F. Solano, 1989: Occurrence of melanin granules and melanosynthesis in the kidney of Sparus auratus. Pigment Cell Res. 2, 93–99. Zuasti, A., C. Ferrer, P. Aroca, and F. Solano, 1990: Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus and Dicentrarchus labrax (Pisces, Teleostei). Pigment Cell Res. 3, 126–131.
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