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Caveolin-3 expression during early chicken development
Developmental Brain Research 141 (2003) 83–89 www.elsevier.com / locate / devbrainres
Research report
Caveolin-3 expression during early chicken development Dong Hoon Shin a , Jin-Soo Kim b , Bum Sun Kwon c , Kun-Sei Lee d , Jong Wan Kim b , Myung Ho Kim c , Sa Sun Cho a , Wang Jae Lee a , * a
Department of Anatomy, Seoul National University, Seoul, South Korea Department of Rehabilitation Medicine, Dankook University, Chonan, South Korea c Department of Orthopaedic Surgery, Dankook University, Chonan, South Korea d Department of Preventive Medicine, Konkuk University College of Medicine, Chungju-City, South Korea b
Accepted 3 December 2002
Abstract Caveolin-3, a protein that is correlated with caveolae, is found in muscle cells, especially during their differentiation. Although the distribution of caveolin-3 has been studied in cases such as adult and late embryonic mammalians, the expression of caveolin-3 has not been clearly defined during chicken development. In this study, we detected intense caveolin-3 immunoreactivity (IR) as early as embryonic day 4 (E4), most of the signals were localized within the neural tube and myotome. While IRs in the brain occurred in radial glia at E6, these intensities were reduced to an almost undetectable level at E8. In the case of muscle cells, the exclusive localization of caveolin-3 in the cytoplasmic membrane was detected even at E11, much earlier than in mammalian muscle tissues. Although the caveolin-3 IR pattern was similar to that reported by previous studies, we found some interesting mismatches in the case of avian tissues. Although we are unable to explain caveolin-3 expression patterns in the early embryonic stages, this study could provide a basis for further study on the function of caveolin-3 in avian embryogenesis. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Genesis of neurons and glia Keywords: Caveolin-3; Muscle; Neural tube; Brain; Immunocytochemistry
1. Introduction A caveola is defined as an invagination of the plasma membrane [17], which is profusely detected in endothelial cells, adipocytes, and muscle cells. In terms of the function of caveolae, several possibilities have been proposed; transport across the endothelial monolayer [9,20], endocytic uptake pathways [1], calcium homeostasis [7,8], and even signal transduction [14]. Caveolin, 21 kD integral membrane protein, was shown to be the major constituent of caveolae, which create
*Corresponding author. Department of Anatomy, Seoul National University College of Medicine, Yongon-Dong, Chongno-Gu, Seoul 110799, South Korea. Tel.: 182-2-740-8208; fax: 82-2-745-9528. E-mail address: [email protected] (W.J. Lee).
caveolar invaginations [5,12,15–17]. For three different caveolin proteins, caveolin-1, -2 and -3 [19,21,22], these caveolins may be correlated with the development of some types of cells [13]. For example, the expression of a muscle-specific caveolin-3 seemed to correlate with muscle differentiation [21,22]. Traditionally, caveolae are known to play a role in the formation of the transverse (T)-tubule system [2,11] during the differentiation of muscle cells. These findings were supported by some morphological evidence, which demonstrated that T-tubules are formed from the repeated budding of caveolae. During the initial stages of T-tubule formation, the caveolae show as short, beaded, tubular structures, which subsequently develop into extensive, three-dimensional networks showing complicated arrays of caveolae [2,11]. Although these studies only show the correlation between T-tubule formation and caveolae, recent studies have also demonstrated that
0165-3806 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0165-3806(02)00645-4
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caveolin-3 might play a role in the T-tubule formation of muscle cells [18]. Although previous studies successfully showed the correlation between caveolin-3 and muscle cell differentiation, no reports have been issued on the expression of caveolin-3 during early embryogenesis. Therefore, we tried to determine the pattern of caveolin-3 IRs in the early embryonic chicken using immunocytochemistry. As we believe that this chicken embryo is an efficient model for development study, we suggest that it could be the basis for further study on the function of caveolin-3 during early embryogenesis.
3. Results
3.1. Caveolin-3 at E4
2. Materials and methods
We examined the caveolin-3 immunoreactivities (IRs) in the whole body section at the E4 stage. Caveolin-3 was found to be intensely expressed in the neural tube, spinal ganglion and myotome with lower intensity in the dermatome and the notochord (Fig. 1A–C). The magnified image, Fig. 1B showed more intense IRs in the peripheral region of the neural tube rather than in the region adjacent to the lumen, suggesting that the caveolin-3 IR was more intensely expressed by differentiating neuroblasts than by dividing neuroepithelial cells. In Fig. 1C, caveolin-3 IR was well localized in the myotome region while the dermatome only showed weaker IRs.
2.1. Animals, antisera and tissue preparation
3.2. Caveolin-3 at E6
The animals used in this experiment were treated in accordance with ‘The Guide for the Care and Use of Laboratory Animals’ (NIH publication No. 86-23, 1985 edition). Embryonic day 4 (E4), 6 (E6), 8 (E8), 11 (E11), and 16 (E16), and postnatal day 6 (P6) white leghorn chickens were used in this study. Fertilized eggs were incubated at 38 8C in a humidified chamber. Postnatal animals were deeply anesthetized with ether and perfused through the heart with 250–1000 ml of 4% (w / v) paraformaldehyde fixative, which was initiated with 200 ml of saline. Tissues were removed 1 h after the perfusion and placed overnight in the same fixative at 4 8C. The tissues were then washed three times in cold PB (0.1 M sodium phosphate buffer, pH 7.6) and cryoprotected by sucrose infiltration, first with 10% sucrose for 2–3 h and then with 30% sucrose overnight. They were then embedded in OCT compound and frozen rapidly in 2-methylbutane precooled to its freezing point with liquid nitrogen. Tissue specimens were cut into 10 mm slices on a Reichert Jung Frigocut cryostat. Sections were thaw-mounted on gelatincoated microscopic slides and stored at 270 8C until required.
2.2. Immunocytochemistry The primary antibody used was a mouse antiserum directed against rat muscle caveolin-3 (Transduction Laboratories). Immunocytochemical staining was performed by utilizing the streptavidin-conjugated Cy-3 staining method. Briefly, the sections were incubated sequentially in (1) primary antiserum diluted 1:1000 overnight at 4 8C, (2) biotinylated anti-mouse IgG (Vector Laboratories) diluted 1:200 for 1 h at room temperature and (3) streptavidin-conjugated Cy3 (Jackson Immunoresearch) diluted 1:500 for 1 h at room temperature. Sections were observed under a Zeiss Axioplot microscope.
At stage E6, we found intense caveolin-3 IRs in the major portion of the brain (Fig. 1D). In the magnified image, the IR structures were arranged in a direction perpendicular to the surface of the ventricular wall (inset of Fig. 1D). In regions external to the brain, the most intense IRs were localized within muscle forming regions, which showed much more intense IRs than the brain region (Fig. 1E). The pattern of caveolin-3 IRs was clearly observed in the vertebral bodies in which caveolin-3 expressing muscle fibers were aligned in rows (Fig. 1F). In magnified images, muscle cells became much more elongated than in the previous E4 stage and seemed to express caveolin-3 IR both in the cytoplasmic membrane and in the cytoplasm (Fig. 1F).
3.3. Caveolin-3 at E8 At E8, IRs in the central nervous system had decreased to the almost undetectable level. On the other hand, muscle cells showed intense caveolin-3 IRs throughout the whole chicken embryo (Fig. 2A). In the magnified image, caveolin-3 IRs were found to be localized mainly within the cytoplasmic membrane, not within the cytoplasm (Fig. 2B). The magnified image of the brain also showed caveolin-3 IRs in a linear pattern running from the ventricular lumen to the outer periphery. Judging from their morphologies, they might have been the radial glia, which are known to play a role as a scaffold in the migration of neural cells (Fig. 2C).
3.4. Caveolin-3 at E11 Most of the caveolin-3 IRs were not localized in the tissues other than muscles (Fig. 2D). In the cross and longitudinal sections shown by Fig. 2E and F, it was clearly observed that the IRs were localized exclusively within the cytoplasmic membrane, showing ring-shaped immunoreactivities (Fig. 2E and F). On the other hand,
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Fig. 1. (A–C) Caveolin-3 immunoreactivities (IRs) at E4. (A) In the neural tube (N), spinal ganglion (S) and myotome (M), intense caveolin-3 IRs were detected with lower intensity in the dermatome (D). (B) Magnified image of the neural tube in A. Note the IRs at the periphery (asterisks) are much more intense than in the region (arrows) adjacent to the lumen (L). (C) Magnified image of the myotome and dermatome in B. The IRs in the myotome were distributed evenly both in the cytoplasmic membrane and cytoplasm. (D–F) Caveolin-3 IRs at E6. (D) Caveolin-3 IRs were detected in the brain. Neuroepithelial cell layer, NE; ventricle, V. Inset showed the magnified image of caveolin-3 IR in the brain region. (E) Very intense IRs were detected in developing muscle cells (asterisks). (F) A remarkable pattern of caveolin-3 IR was found in the vertebral region. Intermittent rows of muscle cells (M) were observed. The inset shows a magnified image of caveolin-3 IR in muscle cells. Note the muscle cells show caveolin-3 expression both in the cytoplasmic membrane and in the cytoplasm. Scale bars5100 mm.
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Fig. 2. (A–C) Caveolin-3 IRs at E8. (A) Cross-section. Note that the IRs in the spinal cord (SP) are much weaker than those in the surrounding muscle fibers (M). C means cartilage. (B) Magnified image of the muscle cells in A. An asymmetrical distribution of caveolin-3 IR in muscle cell is apparent. As intense IRs were mainly localized within the cytoplasmic membrane, not in the cytoplasm, they showed a ring-shaped pattern in cross-section. (C) Magnified image of the spinal cord. Note the caveolin-3 IR (arrows), running from the lumen to the surface of the spinal cord. (D–G) Caveolin-3 IR at E11. (D) Caveolin-3 IR in muscle cells. P, perimysium; E, epimysium. (E and F) Magnified images of the cross (E) and longitudinal (F) sections of muscle cells. Intense caveolin-3 IR (arrows) was only observed within the cytoplasmic membrane. (G) While the brain (Br) showed fewer caveolin-3 IRs, adjacent muscle cells (M) expressed intense IR. (H) Magnified images of the caveolin-3 IR muscle cells which were cross-sectioned. Arrows indicate the exclusive localization of caveolin-3 IRs in the cytoplasmic membrane of muscle fibers. (I) Magnified image of the brain. There were some kinds of the cells which showed caveolin-3 IRs. (J) Cerebellum. Short processes in this region showed caveolin-3 IRs. (K) Magnified image of (J). Both short processes (arrow) and the cells (grey arrow) exhibited caveolin-3 IRs. Scale bars5200 mm.
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Fig. 2. (continued)
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caveolin-3 IR was weakly detected in the central nervous system at this stage (Fig. 2G). While caveolin-3 IRs were intensely observed in the muscle fibers (Fig. 2H), on the magnified image, central nervous system still exhibited caveolin-3 IRs even at this stage (Fig. 2I–K). In various brain regions, some neural cells showed caveolin-3 IRs (Fig. 2I). Most remarkable IRs were also found within the developing cerebellum. Short processes and the cells in the cerebellum showed caveolin-3 (Fig. 2J, K).
4. Discussion
4.1. Previous studies on caveolin-3 expression during muscle development The T-tubule system is known to be an extensive membranous system similar to the muscle plasma membrane even though the system is found more deeply and separately in muscle fiber. Caveolin-3, a general regulator of the plasma membrane and the generator of caveolae, was suggested to be involved with T-tubule system formation during muscle fiber development. Although caveolin-3 might be involved in the initial process of generating the T-tubule domain, the expression of caveolin-3 was restricted to sarcolemmal caveolae, and no longer detectable in the T-tubule system in the case of mature muscle [18]. Briefly, caveolin-3 expression was found to be mainly localized during the late embryonic stages of E16 to 3 days after birth. Immunofluorescence study showed that mouse leg muscle at E16 had strong caveolin-3 IRs around the rims of muscle fibers with some apparent punctuate immunoreactivities throughout crosssectioned muscle cells. Though these IRs were found within the cytoplasm of 18-day embryonic muscle cells, 3-day-old mice showed less IR within the cytoplasm, which showed ring-shaped IRs in muscle cells on crosssection [18]. The association between caveolin-3 and the T-tubule system in differentiating muscle cells was further supported by the finding that caveolin-3 was only localized in precursor T-tubules and not in the mature T-tubule system [3,4,6,18].
4.2. Caveolin-3 expression in the chicken embryo We chose to perform a study on caveolin-3 IR in developing chicken tissues, which has not been previously undertaken. In the present study, caveolin-3 IRs showed some differences as compared with previous studies, even though the general expression pattern is similar to that found in the previous mammalian case [18]. Although IR was found in chicken muscle cells, as was found in mammalian muscles, IR appeared at a much earlier stage in the chicken. The exclusive distribution of caveolin-3 IR in the cytoplasmic membrane started at E8 in the chicken. And before E6, signals were found not only in the
cytoplasmic membrane but also in the cytoplasm, and were evenly distributed throughout the muscle cell fiber. Although we do not know whether these differences are due to differences in the development stages of the two species, it cannot be denied that a similar caveolin-3 IR pattern was found much earlier in the chicken. In addition, at the earliest stage of E4, caveolin-3 IRs were found in some primitive myotome structures, which might have evolved into muscle cells in the adult stage. The differences in signal intensity between the myotome and the dermatome seemed to be important because even at these earliest stages, structures correlated with muscle formation showed intense caveolin-3 IR. Though the function of caveolin-3 at this stage could be quite different from that in the later stages, these findings suggest that caveolin-3 might play a role in some muscle-related function even during this early development period.
4.3. Caveolin-3 in early central nervous system development Traditionally, caveolin-3 expression is believed to be restricted to muscle cells, and caveolins-1 and -2 to be more widely expressed. However, some recent observations have shown that caveolin-3 might be expressed even in brain cells under normal or pathological states. For example, in a study on the involvement of caveolin-3 in the pathophysiology of Alzheimer’s disease (AD), astroglia surrounding senile plaques exhibited a dramatic upregulation of caveolin-3 IR. Although this finding could be taken to demonstrate the presence of caveolin-3 IR only in the pathological brain tissue, another study [10] also reported that caveolin-3 was successfully purified and characterized from brain tissue under non-denaturing conditions [10]. Using ion trap mass spectrometry, they showed the presence of caveolin-3 IR in brain cells, especially in astrocytes. Electron microscopic analysis was also used to show that astrocytes possess numerous caveolar invaginations of the plasma membrane, thus supporting the presence of caveolin [10]. Although this finding was somewhat surprising because previous studies had suggested that the expression of caveolin-3 was confined to striated and smooth muscle cells. In the present study, caveolin-3 expression was detected in the central nervous system during the early development stages in the chicken. At E8, though the signal intensities were relatively low compared with those seen in muscle cell fibers, some structures in the brain showed remarkable caveolin-3 IR. Moreover, even though previous studies found that caveolin-3 IR was only detected in the adult stage, the present study showed that caveolin-3 IR is detectable even during the early embryonic stages. While IR intensities in brain and muscle cells were equally intense at E4 to E6, E8 and E11 tissues showed remarkably reduced caveolin-3 IRs in brain versus muscle cells. In terms of the neural cell types showing caveolin-3
D.H. Shin et al. / Developmental Brain Research 141 (2003) 83–89
IR, the neural tube during its earliest stages and the radial glia at E8 demonstrated remarkable IRs. Considering those studies that have reported caveolin-3 IRs in neuroglia, such as astrocytes, it might be suggested, based on the findings of the present study, that the caveolin-3 IR was exclusively localized within a certain type of glial cell, that is, radial glia at earlier stages. On the other hand, at the late stage such as E11, caveolin-3 IRs were found in some types of the neural cells as well. Although we could not unequivocally determine the function of caveolin-3 during the early embryonic stages, and the meaning of differences in caveolin-3 expression in mammals and birds, we hope that this study provides a basis for the further study of caveolin-3 function in avian embryogenesis.
References [1] R.G.W. Anderson, Potocytosis of small molecules and ions by caveolae, Trends Cell Biol. 3 (1993) 69–72. [2] E.B. Ezerman, H. Ishikawa, Differentiation of the sarcoplasmic reticulum and T-system in developing chick skeletal muscle in vitro, J. Cell Biol. 35 (1967) 405–420. [3] B.E. Flucher, Structural analysis of muscle development: transverse tubules, sarcoplasmic reticulum, and the triad, Dev. Biol. 154 (1992) 245–260. [4] B.E. Flucher, J.L. Phillips, J.A. Powell, S.B. Andrews, M.P. Daniels, Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle in vivo and in vitro, Dev. Biol. 150 (1992) 266–280. [5] A.M. Fra, M. Masserini, P. Palestini, S. Sonnino, K. Simons, A photo-reactive derivative of ganglioside GM1 specifically crosslinks VIP21 caveolin on the cell surface, FEBS Lett. 375 (1995) 11–14. [6] C. Franzini-Armstrong, Simultaneous maturation of transverse tubules and sarcoplasmic reticulum during muscle differentiation in the mouse, Dev. Biol. 146 (1991) 353–362. [7] T. Fujimoto, S. Nakade, A. Miyawaki, K. Mikoshiba, K. Ogawa, Localization of inositol 1,4,5-triphosphate receptor-like protein in plasmalemmal caveolae, J. Cell Biol. 119 (1992) 1507–1513. [8] T. Fujimoto, Calcium pump of the plasma membrane is localized in caveolae, J. Cell Biol. 120 (1993) 1147–1157.
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[9] L. Ghitescu, A. Fixman, M. Simionescu, N. Simionescu, Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis, J. Cell Biol. 102 (1986) 1304–1311. [10] T. Ikezu, H. Ueda, B.D. Trapp, K. Nishiyama, J.F. Sha, D. Volonte, F. Galbiati, A.L. Byrd, G. Bassell, H. Serizawa, W.S. Lane, M.P. Lisanti, T. Okamoto, Affinity-purification and characterization of caveolins from the brain: differential expression of caveolin-1, -2, and -3 in brain endothelial and astroglial cell types, Brain Res. 804 (1998) 177–192. [11] H. Ishikawa, Formation of elaborate networks of T-system tubules in cultured skeletal muscle with special reference to the T-system formation, J. Cell Biol. 38 (1968) 51–66. [12] T.V. Kurzchalia, P. Dupree, S. Monier, VIP21-Caveolin, a protein of the trans-Golgi network and caveolae, FEBS Lett. 346 (1994) 88–91. [13] S. Li, T. Okamoto, M. Chun, M. Sargiacomo, J.E. Casanova, S.H. Hansen, I. Nishimoto, M.P. Lisanti, Evidence for a regulated interaction between heterotrimeric G proteins and caveolin, J. Biol. Chem. 270 (1995) 15693–15701. [14] M.P. Lisanti, P.E. Scherer, Z.-L. Tang, M. Sargiacomo, Caveolae, caveolin and caveolin-rich membrane domains: a signaling hypothesis, Trends Cell Biol. 4 (1994) 231–235. [15] M. Murata, T. Kurzchalia, J. Peranen, R. Schreiner, F.T. Wieland, T. Kurzchalia, K. Simons, VIP21-caveolin is a cholesterol-binding protein, Proc. Natl. Acad. Sci. USA 92 (1995) 10339–10343. [16] R.G. Parton, K. Simons, Digging into caveolae, Science 269 (1995) 1398–1399. [17] R.G. Parton, Caveolae and caveolins, Curr. Opin. Cell Biol. 8 (1996) 542–548. [18] R.G. Parton, M. Way, N. Zorzi, E. Stang, Caveolin-3 associates with developing T-tubules during muscle differentiation, J. Cell Biol. 136 (1997) 137–154. [19] P.E. Scherer, T. Okamoto, M. Chun, H.F. Lodish, M.P. Lisanti, Identification, sequence, and expression of caveolin-2 defines a caveolin gene family, Proc. Natl. Acad. Sci. USA 93 (1996) 131– 135. [20] J.E. Schnitzer, P. Oh, E. Pinney, J. Allard, Filipin-sensitive caveolae mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules, J. Cell Biol. 127 (1994) 1217–1232. [21] Z. Tang, P.E. Scherer, T. Okamoto, K. Song, C. Chu, D.S. Kohtz, I. Nishimoto, H.F. Lodish, M.P. Lisanti, Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle, J. Biol. Chem. 271 (1996) 2255–2261. [22] M. Way, R.G. Parton, M-caveolin, a muscle-specific caveolin-related protein, FEBS Lett. 376 (1995) 108–112.
Research report
Caveolin-3 expression during early chicken development Dong Hoon Shin a , Jin-Soo Kim b , Bum Sun Kwon c , Kun-Sei Lee d , Jong Wan Kim b , Myung Ho Kim c , Sa Sun Cho a , Wang Jae Lee a , * a
Department of Anatomy, Seoul National University, Seoul, South Korea Department of Rehabilitation Medicine, Dankook University, Chonan, South Korea c Department of Orthopaedic Surgery, Dankook University, Chonan, South Korea d Department of Preventive Medicine, Konkuk University College of Medicine, Chungju-City, South Korea b
Accepted 3 December 2002
Abstract Caveolin-3, a protein that is correlated with caveolae, is found in muscle cells, especially during their differentiation. Although the distribution of caveolin-3 has been studied in cases such as adult and late embryonic mammalians, the expression of caveolin-3 has not been clearly defined during chicken development. In this study, we detected intense caveolin-3 immunoreactivity (IR) as early as embryonic day 4 (E4), most of the signals were localized within the neural tube and myotome. While IRs in the brain occurred in radial glia at E6, these intensities were reduced to an almost undetectable level at E8. In the case of muscle cells, the exclusive localization of caveolin-3 in the cytoplasmic membrane was detected even at E11, much earlier than in mammalian muscle tissues. Although the caveolin-3 IR pattern was similar to that reported by previous studies, we found some interesting mismatches in the case of avian tissues. Although we are unable to explain caveolin-3 expression patterns in the early embryonic stages, this study could provide a basis for further study on the function of caveolin-3 in avian embryogenesis. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Genesis of neurons and glia Keywords: Caveolin-3; Muscle; Neural tube; Brain; Immunocytochemistry
1. Introduction A caveola is defined as an invagination of the plasma membrane [17], which is profusely detected in endothelial cells, adipocytes, and muscle cells. In terms of the function of caveolae, several possibilities have been proposed; transport across the endothelial monolayer [9,20], endocytic uptake pathways [1], calcium homeostasis [7,8], and even signal transduction [14]. Caveolin, 21 kD integral membrane protein, was shown to be the major constituent of caveolae, which create
*Corresponding author. Department of Anatomy, Seoul National University College of Medicine, Yongon-Dong, Chongno-Gu, Seoul 110799, South Korea. Tel.: 182-2-740-8208; fax: 82-2-745-9528. E-mail address: [email protected] (W.J. Lee).
caveolar invaginations [5,12,15–17]. For three different caveolin proteins, caveolin-1, -2 and -3 [19,21,22], these caveolins may be correlated with the development of some types of cells [13]. For example, the expression of a muscle-specific caveolin-3 seemed to correlate with muscle differentiation [21,22]. Traditionally, caveolae are known to play a role in the formation of the transverse (T)-tubule system [2,11] during the differentiation of muscle cells. These findings were supported by some morphological evidence, which demonstrated that T-tubules are formed from the repeated budding of caveolae. During the initial stages of T-tubule formation, the caveolae show as short, beaded, tubular structures, which subsequently develop into extensive, three-dimensional networks showing complicated arrays of caveolae [2,11]. Although these studies only show the correlation between T-tubule formation and caveolae, recent studies have also demonstrated that
0165-3806 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0165-3806(02)00645-4
84
D.H. Shin et al. / Developmental Brain Research 141 (2003) 83–89
caveolin-3 might play a role in the T-tubule formation of muscle cells [18]. Although previous studies successfully showed the correlation between caveolin-3 and muscle cell differentiation, no reports have been issued on the expression of caveolin-3 during early embryogenesis. Therefore, we tried to determine the pattern of caveolin-3 IRs in the early embryonic chicken using immunocytochemistry. As we believe that this chicken embryo is an efficient model for development study, we suggest that it could be the basis for further study on the function of caveolin-3 during early embryogenesis.
3. Results
3.1. Caveolin-3 at E4
2. Materials and methods
We examined the caveolin-3 immunoreactivities (IRs) in the whole body section at the E4 stage. Caveolin-3 was found to be intensely expressed in the neural tube, spinal ganglion and myotome with lower intensity in the dermatome and the notochord (Fig. 1A–C). The magnified image, Fig. 1B showed more intense IRs in the peripheral region of the neural tube rather than in the region adjacent to the lumen, suggesting that the caveolin-3 IR was more intensely expressed by differentiating neuroblasts than by dividing neuroepithelial cells. In Fig. 1C, caveolin-3 IR was well localized in the myotome region while the dermatome only showed weaker IRs.
2.1. Animals, antisera and tissue preparation
3.2. Caveolin-3 at E6
The animals used in this experiment were treated in accordance with ‘The Guide for the Care and Use of Laboratory Animals’ (NIH publication No. 86-23, 1985 edition). Embryonic day 4 (E4), 6 (E6), 8 (E8), 11 (E11), and 16 (E16), and postnatal day 6 (P6) white leghorn chickens were used in this study. Fertilized eggs were incubated at 38 8C in a humidified chamber. Postnatal animals were deeply anesthetized with ether and perfused through the heart with 250–1000 ml of 4% (w / v) paraformaldehyde fixative, which was initiated with 200 ml of saline. Tissues were removed 1 h after the perfusion and placed overnight in the same fixative at 4 8C. The tissues were then washed three times in cold PB (0.1 M sodium phosphate buffer, pH 7.6) and cryoprotected by sucrose infiltration, first with 10% sucrose for 2–3 h and then with 30% sucrose overnight. They were then embedded in OCT compound and frozen rapidly in 2-methylbutane precooled to its freezing point with liquid nitrogen. Tissue specimens were cut into 10 mm slices on a Reichert Jung Frigocut cryostat. Sections were thaw-mounted on gelatincoated microscopic slides and stored at 270 8C until required.
2.2. Immunocytochemistry The primary antibody used was a mouse antiserum directed against rat muscle caveolin-3 (Transduction Laboratories). Immunocytochemical staining was performed by utilizing the streptavidin-conjugated Cy-3 staining method. Briefly, the sections were incubated sequentially in (1) primary antiserum diluted 1:1000 overnight at 4 8C, (2) biotinylated anti-mouse IgG (Vector Laboratories) diluted 1:200 for 1 h at room temperature and (3) streptavidin-conjugated Cy3 (Jackson Immunoresearch) diluted 1:500 for 1 h at room temperature. Sections were observed under a Zeiss Axioplot microscope.
At stage E6, we found intense caveolin-3 IRs in the major portion of the brain (Fig. 1D). In the magnified image, the IR structures were arranged in a direction perpendicular to the surface of the ventricular wall (inset of Fig. 1D). In regions external to the brain, the most intense IRs were localized within muscle forming regions, which showed much more intense IRs than the brain region (Fig. 1E). The pattern of caveolin-3 IRs was clearly observed in the vertebral bodies in which caveolin-3 expressing muscle fibers were aligned in rows (Fig. 1F). In magnified images, muscle cells became much more elongated than in the previous E4 stage and seemed to express caveolin-3 IR both in the cytoplasmic membrane and in the cytoplasm (Fig. 1F).
3.3. Caveolin-3 at E8 At E8, IRs in the central nervous system had decreased to the almost undetectable level. On the other hand, muscle cells showed intense caveolin-3 IRs throughout the whole chicken embryo (Fig. 2A). In the magnified image, caveolin-3 IRs were found to be localized mainly within the cytoplasmic membrane, not within the cytoplasm (Fig. 2B). The magnified image of the brain also showed caveolin-3 IRs in a linear pattern running from the ventricular lumen to the outer periphery. Judging from their morphologies, they might have been the radial glia, which are known to play a role as a scaffold in the migration of neural cells (Fig. 2C).
3.4. Caveolin-3 at E11 Most of the caveolin-3 IRs were not localized in the tissues other than muscles (Fig. 2D). In the cross and longitudinal sections shown by Fig. 2E and F, it was clearly observed that the IRs were localized exclusively within the cytoplasmic membrane, showing ring-shaped immunoreactivities (Fig. 2E and F). On the other hand,
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Fig. 1. (A–C) Caveolin-3 immunoreactivities (IRs) at E4. (A) In the neural tube (N), spinal ganglion (S) and myotome (M), intense caveolin-3 IRs were detected with lower intensity in the dermatome (D). (B) Magnified image of the neural tube in A. Note the IRs at the periphery (asterisks) are much more intense than in the region (arrows) adjacent to the lumen (L). (C) Magnified image of the myotome and dermatome in B. The IRs in the myotome were distributed evenly both in the cytoplasmic membrane and cytoplasm. (D–F) Caveolin-3 IRs at E6. (D) Caveolin-3 IRs were detected in the brain. Neuroepithelial cell layer, NE; ventricle, V. Inset showed the magnified image of caveolin-3 IR in the brain region. (E) Very intense IRs were detected in developing muscle cells (asterisks). (F) A remarkable pattern of caveolin-3 IR was found in the vertebral region. Intermittent rows of muscle cells (M) were observed. The inset shows a magnified image of caveolin-3 IR in muscle cells. Note the muscle cells show caveolin-3 expression both in the cytoplasmic membrane and in the cytoplasm. Scale bars5100 mm.
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Fig. 2. (A–C) Caveolin-3 IRs at E8. (A) Cross-section. Note that the IRs in the spinal cord (SP) are much weaker than those in the surrounding muscle fibers (M). C means cartilage. (B) Magnified image of the muscle cells in A. An asymmetrical distribution of caveolin-3 IR in muscle cell is apparent. As intense IRs were mainly localized within the cytoplasmic membrane, not in the cytoplasm, they showed a ring-shaped pattern in cross-section. (C) Magnified image of the spinal cord. Note the caveolin-3 IR (arrows), running from the lumen to the surface of the spinal cord. (D–G) Caveolin-3 IR at E11. (D) Caveolin-3 IR in muscle cells. P, perimysium; E, epimysium. (E and F) Magnified images of the cross (E) and longitudinal (F) sections of muscle cells. Intense caveolin-3 IR (arrows) was only observed within the cytoplasmic membrane. (G) While the brain (Br) showed fewer caveolin-3 IRs, adjacent muscle cells (M) expressed intense IR. (H) Magnified images of the caveolin-3 IR muscle cells which were cross-sectioned. Arrows indicate the exclusive localization of caveolin-3 IRs in the cytoplasmic membrane of muscle fibers. (I) Magnified image of the brain. There were some kinds of the cells which showed caveolin-3 IRs. (J) Cerebellum. Short processes in this region showed caveolin-3 IRs. (K) Magnified image of (J). Both short processes (arrow) and the cells (grey arrow) exhibited caveolin-3 IRs. Scale bars5200 mm.
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Fig. 2. (continued)
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caveolin-3 IR was weakly detected in the central nervous system at this stage (Fig. 2G). While caveolin-3 IRs were intensely observed in the muscle fibers (Fig. 2H), on the magnified image, central nervous system still exhibited caveolin-3 IRs even at this stage (Fig. 2I–K). In various brain regions, some neural cells showed caveolin-3 IRs (Fig. 2I). Most remarkable IRs were also found within the developing cerebellum. Short processes and the cells in the cerebellum showed caveolin-3 (Fig. 2J, K).
4. Discussion
4.1. Previous studies on caveolin-3 expression during muscle development The T-tubule system is known to be an extensive membranous system similar to the muscle plasma membrane even though the system is found more deeply and separately in muscle fiber. Caveolin-3, a general regulator of the plasma membrane and the generator of caveolae, was suggested to be involved with T-tubule system formation during muscle fiber development. Although caveolin-3 might be involved in the initial process of generating the T-tubule domain, the expression of caveolin-3 was restricted to sarcolemmal caveolae, and no longer detectable in the T-tubule system in the case of mature muscle [18]. Briefly, caveolin-3 expression was found to be mainly localized during the late embryonic stages of E16 to 3 days after birth. Immunofluorescence study showed that mouse leg muscle at E16 had strong caveolin-3 IRs around the rims of muscle fibers with some apparent punctuate immunoreactivities throughout crosssectioned muscle cells. Though these IRs were found within the cytoplasm of 18-day embryonic muscle cells, 3-day-old mice showed less IR within the cytoplasm, which showed ring-shaped IRs in muscle cells on crosssection [18]. The association between caveolin-3 and the T-tubule system in differentiating muscle cells was further supported by the finding that caveolin-3 was only localized in precursor T-tubules and not in the mature T-tubule system [3,4,6,18].
4.2. Caveolin-3 expression in the chicken embryo We chose to perform a study on caveolin-3 IR in developing chicken tissues, which has not been previously undertaken. In the present study, caveolin-3 IRs showed some differences as compared with previous studies, even though the general expression pattern is similar to that found in the previous mammalian case [18]. Although IR was found in chicken muscle cells, as was found in mammalian muscles, IR appeared at a much earlier stage in the chicken. The exclusive distribution of caveolin-3 IR in the cytoplasmic membrane started at E8 in the chicken. And before E6, signals were found not only in the
cytoplasmic membrane but also in the cytoplasm, and were evenly distributed throughout the muscle cell fiber. Although we do not know whether these differences are due to differences in the development stages of the two species, it cannot be denied that a similar caveolin-3 IR pattern was found much earlier in the chicken. In addition, at the earliest stage of E4, caveolin-3 IRs were found in some primitive myotome structures, which might have evolved into muscle cells in the adult stage. The differences in signal intensity between the myotome and the dermatome seemed to be important because even at these earliest stages, structures correlated with muscle formation showed intense caveolin-3 IR. Though the function of caveolin-3 at this stage could be quite different from that in the later stages, these findings suggest that caveolin-3 might play a role in some muscle-related function even during this early development period.
4.3. Caveolin-3 in early central nervous system development Traditionally, caveolin-3 expression is believed to be restricted to muscle cells, and caveolins-1 and -2 to be more widely expressed. However, some recent observations have shown that caveolin-3 might be expressed even in brain cells under normal or pathological states. For example, in a study on the involvement of caveolin-3 in the pathophysiology of Alzheimer’s disease (AD), astroglia surrounding senile plaques exhibited a dramatic upregulation of caveolin-3 IR. Although this finding could be taken to demonstrate the presence of caveolin-3 IR only in the pathological brain tissue, another study [10] also reported that caveolin-3 was successfully purified and characterized from brain tissue under non-denaturing conditions [10]. Using ion trap mass spectrometry, they showed the presence of caveolin-3 IR in brain cells, especially in astrocytes. Electron microscopic analysis was also used to show that astrocytes possess numerous caveolar invaginations of the plasma membrane, thus supporting the presence of caveolin [10]. Although this finding was somewhat surprising because previous studies had suggested that the expression of caveolin-3 was confined to striated and smooth muscle cells. In the present study, caveolin-3 expression was detected in the central nervous system during the early development stages in the chicken. At E8, though the signal intensities were relatively low compared with those seen in muscle cell fibers, some structures in the brain showed remarkable caveolin-3 IR. Moreover, even though previous studies found that caveolin-3 IR was only detected in the adult stage, the present study showed that caveolin-3 IR is detectable even during the early embryonic stages. While IR intensities in brain and muscle cells were equally intense at E4 to E6, E8 and E11 tissues showed remarkably reduced caveolin-3 IRs in brain versus muscle cells. In terms of the neural cell types showing caveolin-3
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IR, the neural tube during its earliest stages and the radial glia at E8 demonstrated remarkable IRs. Considering those studies that have reported caveolin-3 IRs in neuroglia, such as astrocytes, it might be suggested, based on the findings of the present study, that the caveolin-3 IR was exclusively localized within a certain type of glial cell, that is, radial glia at earlier stages. On the other hand, at the late stage such as E11, caveolin-3 IRs were found in some types of the neural cells as well. Although we could not unequivocally determine the function of caveolin-3 during the early embryonic stages, and the meaning of differences in caveolin-3 expression in mammals and birds, we hope that this study provides a basis for the further study of caveolin-3 function in avian embryogenesis.
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