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Macrophage localization in the developing lens primordium of the mouse embryo – An immunohistochemical study
Experimental Eye Research 83 (2006) 223e228 www.elsevier.com/locate/yexer
Macrophage localization in the developing lens primordium of the mouse embryo e An immunohistochemical study Koji Nishitani*, Kazunobu Sasaki Department of Anatomy, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan Received 15 March 2005; accepted in revised form 7 December 2005 Available online 20 March 2006
Abstract In mammals, macrophages are known to play an important role in lens development. Macrophages in the embryonic lens are positive for F4/80 monoclonal antibody, and, from 10.5 days to 12 days of gestation, numerous macrophages were observed in the ectoderm, lens vesicle, lens cavity and surrounding mesenchymal tissue, phagocytosing and removing degenerating epithelial cells. During primary lens fiber differentiation, the narrowing lens cavity contained numerous macrophages. Most of the macrophages in the cavity attached to the anterior epithelial wall of the lens vesicle, but a few macrophages were found within the lens epithelial cell layer. Conversely, the thickening posterior wall of the vesicle did not contain any positive cells. After the lens cavity was filled, intralental positive cells disappeared. These characteristic localizations of macrophages in the developing lens to remove apoptotic dead cells may indicate that cell death took place mainly in the anterior wall of the lens vesicle, that is, in the lens epithelium. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: lens formation; apoptosis; lens vesicle; macrophage; F4/80
1. Introduction During eye development in mouse embryos, macrophages have been known to be intimately involved in retinal development (Hume et al., 1983; Tripathi et al., 1991), regression of the tunica vasculosa lentis (McMenamin et al., 2002), regression of hyaloid artery (Taniguchi et al., 1999) and regression of the pupillary membrane (Diez-Roux and Lang, 1997). In the development of the lens, macrophages also appear and remove apoptotic dead cells around the lens stalk (GarciaPorrero et al., 1984). Macrophages are one of the cell elements regularly found in normal lens formation and phagocytic cells have been reported to move freely in the lens cavity (Wrenn and Wessells, 1969). However, very little information is available regarding
* Corresponding author. Tel.: þ81 86 462 1199. E-mail address: [email protected] (K. Nishitani). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.12.008
their precise localization in the developing lens primordium and cellular elements in the closing lens cavity. The aim of the present study was to obtain data on the characteristic distribution of macrophages with regard to their possible role in early lens formation from immune histochemical and ultrastructural observations. 2. Materials and methods 2.1. Animals A total of 48 ICR mice were used in this study. One adult male mouse and one adult female mouse were caged together, and next morning was taken as day 0 of pregnancy. Pregnant female mice were killed by cervical dislocation at 10.5, 11.5, 12, 13, 13.5 and 14 days after copulation. On each gestational day, at least four embryos were removed from the uterus under a stereoscopic microscope for histochemical and ultrastructural observations.
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2.2. Immunohistochemical identification of macrophages in developing eyes The embryos were immersed in 4% periodate-lysine paraformaldehyde (PLP), pH 7.4, at 4 C for 12 h, washed with phosphate buffered saline (PBS), dehydrated in graded concentrations of ethanols and embedded in paraffin. Serial 5 mm sections of the heads were made in a frontal plane, and were mounted on glass slides. For immunohistochemistry, sections were incubated with 0.25% Casein (DAKO, Denmark) for 10 min at room temperature, followed by incubation with rat monoclonal antibody against the F4/80 antigen (first antibody, 1:100 dilutions; A3-1, Cosmobio Co., Japan) for 1 h. After washing with PBS, the sections were incubated with horseradish-peroxidase conjugated with goat anti-rat IgG antibody (second antibody, 1:100 dilutions, ICN, USA) for 1 h at room temperature. Staining of F4/80 was specific and not observed when the first antibody was omitted. The HRP-binding sites were detected in 0.005% 3,30 -diamiobenzidine 4HCl and 0.001% H2O2 in 0.1 M TriseHCl buffer. 2.3. Electron microscopic observation Sagittal half heads, resected from the embryos, were immediately cut into 2 mm tissue blocks including eye anlage. The tissue blocks were fixed in 4% paraformaldehyde and 5% glutaraldehyde at 4 C for 3 h and postfixed in 1% osmium at
4 C for 2 h. After dehydration in a series of graded ethanols, the tissue blocks were embedded in Epon 812. Ultrathin sections were cut with an ultramicrotome, stained with uranyl acetate and lead citrate and observed in a JEM-2000EX electron microscope. These experiments were approved by the Animal Research Committee of Kawasaki Medical School (No. 02-095) and conducted according to the ‘‘Guide for the Care and Use of Laboratory Animals’’ of Kawasaki Medical School. 3. Results In mouse lens development, the lens placode appeared as a local thickening of the surface ectoderm at 9.5 days of gestation. At 10.5 days of gestation, the central portion of the lens placode began to invaginate. By day 11.5, the lens vesicle formed its cavity, which was connected with the surface ectoderm by the lens stalk, and then the stalk disappeared completely. From 12 to 14 days of gestation, further differentiation of the lens vesicle led to the formation of the mature lens, composed of two cell types, the epithelial cells of the anterior cell layer and the lens fiber cells of the posterior cell layer. Due to the elongation of lens fiber cells, the hollow lens vesicle was filled at 14 days of gestation. During this early development process of the lens, numerous macrophages appeared not only in the surrounding mesenchyme but also within the lens primordium.
Fig. 1. F4/80 immunohistochemical staining of the lens primordium. Lens placode at (a) 10.5 days of gestation: positive cells (arrows) are present in the peripheral ectoderm of the lens placode and in the space between the lens placode (LP) and the inner layer of the optic cup (IL), X200; (b) 11.5 days of gestation: the arrow head indicates a positive cell in the epithelium of the narrow lens stalk, and arrows indicate positive cells in the lens cavity (*). Mesenchymal tissue (M) around the lens stalk also contains numerous positive cells. Lens vesicle (LV); Inner layer of the optic cup (IL), X230; (c) 12 days of gestation: positive cells (arrows) are present in the lens cavity (*), and a few positive cells (arrow head) are also contained in the anterior wall of the lens vesicle (LV). Inner layer of the optic cup (IL), 160X; (d) 13 days of gestation. Many positive cells (arrows) in the lens cavity (*) make close contact with the posterior surface of the lens epithelium (LE). The primary lens fiber (LF), corresponding to the posterior wall of the lens vesicle, does not contain any positive cells at all. Inner layer of the optic cup (IL), X140; (e) 13.5 days of gestation: arrows indicate many positive cells on both the anterior and posterior surface of the lens epithelium (LE). Numerous positive cells appear in the primitive vitreous cavity (VC), but the developing primary lens fiber (LF) does not contain any positive cells. Inner layer of the optic cup (IL); Lens cavity (*), X90. (f) Lens cavity closure at 14 days of gestation. Positive cells cannot be found within the primitive lens. A positive cell (arrow) may just be observed on the anterior wall of the primitive lens. Cornea (C); Lens epithelium (LE); Primary lens fiber (LF); Vitreous cavity (VC); Inner layer of the optic cup (IL), X60.
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Fig. 2. Electron micrographs of the lens pit and the lens cavity. (a) A low-powered micrograph at 10.5 days of gestation. Numerous dead cells appear in the epithelium of the transitional region from surface ectoderm (EC) to lens placode (LP). Neighboring epithelial cells and macrophages (M) phagocytose them. Lens pit (LPi), scale bar ¼ 10 mm. (b) Epithelium of the framed area in (a). Epithelial cells (E) often appear to release a small round portion of the cytoplasm (arrow) into the lens pit. Detached cell fragments (white arrows) are seen in the lens pit (LPi); scale bar ¼ 2 mm. (c) A low-powered micrograph at 11.5 days of gestation. Numerous cell death fragments (*) are observed in the epithelium of the lens stalk (LS). In the lens cavity (LC), near the anterior wall of the lens vesicle (LV), many round or irregular shaped cell fragments (arrows) could be seen. Surface ectoderm (EC), scale bar ¼ 10 mm. (d) Cell fragments in the lens cavity of the framed area in (c). Cell fragments (white arrows) are generally round, 1.8e2.3 mm in diameter. Lens epithelial cell of the lens vesicle (LE); lens cavity (LC), scale bar ¼ 1 mm.
3.1. Localization of F4/80 positive cells in the lens primordium At around 10 days of gestation, the optic vesicle began to invaginate to form the optic cup, and, at the same time, the lens placode became infolded to form the lens pit. A few positive cells appeared not only in the lumen of the pit but also in the surface ectoderm at the periphery of the lens placode (Fig. 1a). F4/80 positive cells then increased in number, and could be found in the lens stalk, adjacent mesenchymal tissues and the narrow space between the lens vesicle and the optic cup (Fig. 1b). The lens vesicle became detached from the surface
ectoderm at day 11.5, and F4/80 positive cells could be recognized in both the anterior epithelial wall and the lens cavity (Fig. 1c). However, hardly any positive cells at all could be detected in the posterior wall of the lens vesicle (Fig. 1d). During primary lens fiber differentiation around day 13, the narrowing lens cavity contained numerous positive cells. Most of the positive cells in the cavity attached to the anterior epithelial wall of the lens vesicle, but a few positive cells were also found within the lens epithelial cell layer. The thickening posterior wall of the vesicle, on the contrary, contained no positive cells (Fig. 1e). Since positive cells in the space in front of the lens vesicle were also attached to the outer lens surface, the
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Fig. 3. Electron micrographs of the lens vesicle at 11.5 days of gestation. (a) A low-powered micrograph of the lens vesicle. The lens stalk (LS) can be recognized between the surface ectoderm (EC) and the lens vesicle (LV). Macrophages (*) are located in the ectoderm epithelium, lens stalk and lens cavity; scale bar ¼ 10 mm. (b) A mitotic macrophage in the lens cavity of (a). This macrophage has numerous villous projections (arrows) from the cell surface and includes several phagosomes (P) derived from a dying lens epithelial cell; scale bar ¼ 2 mm. (c) A macrophage in the lens epithelium of (a). This macrophage (Mp) includes a large phagosome derived from an epithelial cell having a condensed nucleus (arrow); scale bar ¼ 2 mm. (d) A detached epithelial cell in the lens cavity. A detached epithelial cell (DE) in lens cavity (LC) retains a junctional association (arrow) with an anterior lens wall cell; scale bar ¼ 2 mm.
epithelial layer appeared to be sandwiched by numerous macrophages (Fig. 1e). After the lens cavity was filled, positive cells disappeared, and thereafter the lens did not contain any F4/80 positive cells at all. Positive cells were observed not only in the mesenchyme between the cornea and the lens
but also in the primitive vitreous cavity between the lens and the prospective retina (Fig. 1f). The number of F4/80 positive cells in the primary vitreous cavity behind the lens appeared to be markedly increased after 14 days of gestation (Fig. 1d, e).
Fig. 4. Electron micrographs of the lens vesicle at 13.5 days of gestation. (a) A low-powered micrograph of the lens cavity. Arrows indicate macrophages in the lens cavity (LC). Lens epithelium (LE), scale bar ¼ 10 mm. (b) A macrophage in the lens cavity of (a). This macrophage has numerous projections on the cell surface and includes numerous vacuoles (*) in the cytoplasm. The arrow indicates cell contacts between the macrophage and epithelial cells of the anterior wall of the lens vesicle; scale bar ¼ 2 mm.
K. Nishitani, K. Sasaki / Experimental Eye Research 83 (2006) 223e228
3.2. Ultrastructure of macrophages in the lens primordium At 10.5 days of gestation, the lens placode become indented, and then the lens pit was formed. In the epithelium at the margin of the lens pit, numerous dying cells appeared, and the dead cells were engulfed in the epithelium by neighboring epithelial cells and scavenger macrophages (Fig. 2a). Some epithelial cells appeared to release small cytoplasmic fragments into the lens pit (Fig. 2b). At the 11.5 days of gestation, the lens pit was closed to form the lens cavity where many small cell fragments could be observed. The fragments were characteristically located near the anterior wall of the lens vesicle (Fig. 2c, d). The lens cavity contained two different kinds of cellular elements; scavenger macrophages and fragments from epithelial cells (Fig. 3). At 12 days of gestation, when the lens stalk disappeared, the round lens vesicle separated from the surface ectoderm. Both the lens vesicle and the mesenchyme between the surface ectoderm and the lens vesicle contained numerous macrophages. The cytoplasm held phagosomes derived from dying epithelial cell debris and cell fragments (Fig. 3a). Macrophages in mitosis were often distributed in the lens cavity (Fig. 3b). Macrophages in the lens epithelium contained the dying epithelial cells with nuclei composed of highly condensed chromatin (Fig. 3c), a typical sign of apoptosis. The lens cavity also sometimes contained epithelial cells which had detached of the lens anterior wall (Fig. 3d). At 13.5 days of gestation, macrophages were often seen making intimate contact with the posterior surface of the lens epithelium in the cavity (Fig. 4a). The macrophages had very large vacuoles in their cytoplasm, and often attached to the surface of lens epithelial cells, and were characterized by fingerlike projections (Fig. 4b).
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origins; scavenger macrophages, dying cells and cell components derived from epithelial cells. In the closed space, not neighboring epithelial cells but scavenger macrophages could be responsible for the removal of cellular elements which fall out of the epithelium. Our observation also showed that macrophages displayed a characteristic localization in the space of the lens cavity. Studies on lens development have demonstrated that the lens cavity in the lens vesicle is fated to disappear due to elongation of the primary lens fiber (Davidson, 1981). During the closing process, epithelial cells undergoing apoptosis were frequently observed in the epithelial stalk, adjacent ectoderm and anterior wall of the lens vesicle (Mohamed and Amemiya, 2003). As shown in our results, apoptotic cells, showing chromatin agglutination, were present in the lens epithelium but not in the primary lens fiber at this stage. At the same time, the epithelium began to be sandwiched by numerous F4/80 positive cells, although positive cells could not be found at all in the posterior wall consisting of primary lens fiber. During the process of apoptosis in mammalian systems, functional detail concerning the phagocytosis of apoptotic cells by macrophages was elucidated (Potten and Wilson, 2004). Macrophages are known to recognize specific changes in dying cells, and engulf them. Then the macrophages are stimulated to produce a signaling molecule of transforming growth factor-b1. The most important functional significance of lens macrophages might be as a scavenger for the removal of either apoptotic cells or cell fragments. The characteristic localization of macrophages in developing lens could be considered to reflect the occurrence of apoptosis in the developing lens. The physiological significance of cytoplasmic fragments of epithelial cells in the lens cavity should possibly be considered with regard to the developmental change in the lens epithelial cells.
4. Discussion
Acknowledgements
The present study clearly showed that scavenger macrophages are intimately involved in early lens formation. As mentioned above, numerous F4/80 positive cells were observed in the ectoderm, lens vesicle, lens cavity and surrounding mesenchymal tissue from 10.5 days to 12 days of gestation. These F4/80 positive cells could be identified as macrophages by their ultrastructural features, and they often held large phagosomes probably derived from either dying epithelial cells or cell fragments. At the early stage of lens formation, cell death by apoptosis has previously been observed in the lens primordium (Wride, 1996; Laemle et al., 1999; Mohamed and Amemiya, 2003). Our results showed that, in addition to the neighboring epithelial cells, macrophages appeared in the epithelium at the border of the lens placode, and removed these dead cells. Pei and Rhodin (1970) reported that, within the pit and cavity of the lens vesicle, degenerating cells could be found, and, in addition to the dying cells, cytoplasmic fragments of epithelial cells also appeared to fall into either the lens pit or the lens cavity. In the cavity of the lens vesicle, therefore, there exist several cellular elements from multiple
The authors wish to thank Mr. K. Uehira, Mr. T. Suda and Mrs. M. Suda for their skillful technical assistance. This work was supported in part by a research project grant from Kawasaki Medical School (140-201,2002).
Reference Davidson, H., 1981. The lens. In: Davidson, H. (Ed.), Physiology of the Eye. MacMillan Press, pp. 139e201. Diez-Roux, G., Lang, R.A., 1997. Macrophages induce apoptosis in normal cells in vivo. Development. 124, 3633e3638. Garcia-Porrero, J.A., Colvee, E., Ojeda, J., 1984. The mechanisms of cell death and phagocytosis in the early chick lens morphogenesis: a scanning electron microscopy and cytochemical approach. Anat. Rec. 208, 123e136. Hume, D.A., Perry, V.H., Gordon, S., 1983. Immunohistochemical localization of a macrophage-specific antigen in developing mouse retina: phagocytosis of dying neurons and differentiation of microglial cells to form a regular array in the plexiform layers. J. Cell Biol. 97, 253e257. Laemle, L.K., Puszkarczuk, M., Feinberg, R.N., 1999. Apoptosis in early ocular morphogenesis in the mouse. Dev. Brain Res. 112, 129e133.
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McMenamin, P.G., Djano, J., Wealtball, R., Griffin, B.J., 2002. Characterization of the macrophages associated with the tunica vasculosa lentis of the rat eye. Investig. Ophthalmol. Vis. Sci. 43, 2076e2082. Mohamed, Y.H., Amemiya, T., 2003. Apoptosis and lens vesicle development. Eur. J. Ophthalmol. 13, 1e10. Pei, Y.F., Rhodin, J.A.G., 1970. The prenatal development of the mouse eye. Anat. Rec. 168, 105e125. Potten, C., Wilson, J., 2004. What to wear and who clears up the rubbish? Apoptosis. Cambridge University Press, pp. 61e66.
Taniguchi, H., Kitaoka, T., Gong, H., Amemiya, T., 1999. Apoptosis of the hyaloid artery in the rat eye. Ann. Anat. 181, 555e560. Tripathi, B.J., Tripathi, R.C., Livingstone, A.M., Borisuth, N.S.C., 1991. The role of growth factor in the embryogenesis and differentiation of the eye. Am. J. Anat. 192, 442e471. Wrenn, J.T., Wessells, N.K., 1969. An ultrastructural study of lens invagination in the mouse. J. Exp. Zool. 171, 359e367. Wride, M.A., 1996. Cellular and molecular features of lens differentiation: a review of recent advances. Differentiation 61, 77e93.
Macrophage localization in the developing lens primordium of the mouse embryo e An immunohistochemical study Koji Nishitani*, Kazunobu Sasaki Department of Anatomy, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan Received 15 March 2005; accepted in revised form 7 December 2005 Available online 20 March 2006
Abstract In mammals, macrophages are known to play an important role in lens development. Macrophages in the embryonic lens are positive for F4/80 monoclonal antibody, and, from 10.5 days to 12 days of gestation, numerous macrophages were observed in the ectoderm, lens vesicle, lens cavity and surrounding mesenchymal tissue, phagocytosing and removing degenerating epithelial cells. During primary lens fiber differentiation, the narrowing lens cavity contained numerous macrophages. Most of the macrophages in the cavity attached to the anterior epithelial wall of the lens vesicle, but a few macrophages were found within the lens epithelial cell layer. Conversely, the thickening posterior wall of the vesicle did not contain any positive cells. After the lens cavity was filled, intralental positive cells disappeared. These characteristic localizations of macrophages in the developing lens to remove apoptotic dead cells may indicate that cell death took place mainly in the anterior wall of the lens vesicle, that is, in the lens epithelium. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: lens formation; apoptosis; lens vesicle; macrophage; F4/80
1. Introduction During eye development in mouse embryos, macrophages have been known to be intimately involved in retinal development (Hume et al., 1983; Tripathi et al., 1991), regression of the tunica vasculosa lentis (McMenamin et al., 2002), regression of hyaloid artery (Taniguchi et al., 1999) and regression of the pupillary membrane (Diez-Roux and Lang, 1997). In the development of the lens, macrophages also appear and remove apoptotic dead cells around the lens stalk (GarciaPorrero et al., 1984). Macrophages are one of the cell elements regularly found in normal lens formation and phagocytic cells have been reported to move freely in the lens cavity (Wrenn and Wessells, 1969). However, very little information is available regarding
* Corresponding author. Tel.: þ81 86 462 1199. E-mail address: [email protected] (K. Nishitani). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.12.008
their precise localization in the developing lens primordium and cellular elements in the closing lens cavity. The aim of the present study was to obtain data on the characteristic distribution of macrophages with regard to their possible role in early lens formation from immune histochemical and ultrastructural observations. 2. Materials and methods 2.1. Animals A total of 48 ICR mice were used in this study. One adult male mouse and one adult female mouse were caged together, and next morning was taken as day 0 of pregnancy. Pregnant female mice were killed by cervical dislocation at 10.5, 11.5, 12, 13, 13.5 and 14 days after copulation. On each gestational day, at least four embryos were removed from the uterus under a stereoscopic microscope for histochemical and ultrastructural observations.
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2.2. Immunohistochemical identification of macrophages in developing eyes The embryos were immersed in 4% periodate-lysine paraformaldehyde (PLP), pH 7.4, at 4 C for 12 h, washed with phosphate buffered saline (PBS), dehydrated in graded concentrations of ethanols and embedded in paraffin. Serial 5 mm sections of the heads were made in a frontal plane, and were mounted on glass slides. For immunohistochemistry, sections were incubated with 0.25% Casein (DAKO, Denmark) for 10 min at room temperature, followed by incubation with rat monoclonal antibody against the F4/80 antigen (first antibody, 1:100 dilutions; A3-1, Cosmobio Co., Japan) for 1 h. After washing with PBS, the sections were incubated with horseradish-peroxidase conjugated with goat anti-rat IgG antibody (second antibody, 1:100 dilutions, ICN, USA) for 1 h at room temperature. Staining of F4/80 was specific and not observed when the first antibody was omitted. The HRP-binding sites were detected in 0.005% 3,30 -diamiobenzidine 4HCl and 0.001% H2O2 in 0.1 M TriseHCl buffer. 2.3. Electron microscopic observation Sagittal half heads, resected from the embryos, were immediately cut into 2 mm tissue blocks including eye anlage. The tissue blocks were fixed in 4% paraformaldehyde and 5% glutaraldehyde at 4 C for 3 h and postfixed in 1% osmium at
4 C for 2 h. After dehydration in a series of graded ethanols, the tissue blocks were embedded in Epon 812. Ultrathin sections were cut with an ultramicrotome, stained with uranyl acetate and lead citrate and observed in a JEM-2000EX electron microscope. These experiments were approved by the Animal Research Committee of Kawasaki Medical School (No. 02-095) and conducted according to the ‘‘Guide for the Care and Use of Laboratory Animals’’ of Kawasaki Medical School. 3. Results In mouse lens development, the lens placode appeared as a local thickening of the surface ectoderm at 9.5 days of gestation. At 10.5 days of gestation, the central portion of the lens placode began to invaginate. By day 11.5, the lens vesicle formed its cavity, which was connected with the surface ectoderm by the lens stalk, and then the stalk disappeared completely. From 12 to 14 days of gestation, further differentiation of the lens vesicle led to the formation of the mature lens, composed of two cell types, the epithelial cells of the anterior cell layer and the lens fiber cells of the posterior cell layer. Due to the elongation of lens fiber cells, the hollow lens vesicle was filled at 14 days of gestation. During this early development process of the lens, numerous macrophages appeared not only in the surrounding mesenchyme but also within the lens primordium.
Fig. 1. F4/80 immunohistochemical staining of the lens primordium. Lens placode at (a) 10.5 days of gestation: positive cells (arrows) are present in the peripheral ectoderm of the lens placode and in the space between the lens placode (LP) and the inner layer of the optic cup (IL), X200; (b) 11.5 days of gestation: the arrow head indicates a positive cell in the epithelium of the narrow lens stalk, and arrows indicate positive cells in the lens cavity (*). Mesenchymal tissue (M) around the lens stalk also contains numerous positive cells. Lens vesicle (LV); Inner layer of the optic cup (IL), X230; (c) 12 days of gestation: positive cells (arrows) are present in the lens cavity (*), and a few positive cells (arrow head) are also contained in the anterior wall of the lens vesicle (LV). Inner layer of the optic cup (IL), 160X; (d) 13 days of gestation. Many positive cells (arrows) in the lens cavity (*) make close contact with the posterior surface of the lens epithelium (LE). The primary lens fiber (LF), corresponding to the posterior wall of the lens vesicle, does not contain any positive cells at all. Inner layer of the optic cup (IL), X140; (e) 13.5 days of gestation: arrows indicate many positive cells on both the anterior and posterior surface of the lens epithelium (LE). Numerous positive cells appear in the primitive vitreous cavity (VC), but the developing primary lens fiber (LF) does not contain any positive cells. Inner layer of the optic cup (IL); Lens cavity (*), X90. (f) Lens cavity closure at 14 days of gestation. Positive cells cannot be found within the primitive lens. A positive cell (arrow) may just be observed on the anterior wall of the primitive lens. Cornea (C); Lens epithelium (LE); Primary lens fiber (LF); Vitreous cavity (VC); Inner layer of the optic cup (IL), X60.
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Fig. 2. Electron micrographs of the lens pit and the lens cavity. (a) A low-powered micrograph at 10.5 days of gestation. Numerous dead cells appear in the epithelium of the transitional region from surface ectoderm (EC) to lens placode (LP). Neighboring epithelial cells and macrophages (M) phagocytose them. Lens pit (LPi), scale bar ¼ 10 mm. (b) Epithelium of the framed area in (a). Epithelial cells (E) often appear to release a small round portion of the cytoplasm (arrow) into the lens pit. Detached cell fragments (white arrows) are seen in the lens pit (LPi); scale bar ¼ 2 mm. (c) A low-powered micrograph at 11.5 days of gestation. Numerous cell death fragments (*) are observed in the epithelium of the lens stalk (LS). In the lens cavity (LC), near the anterior wall of the lens vesicle (LV), many round or irregular shaped cell fragments (arrows) could be seen. Surface ectoderm (EC), scale bar ¼ 10 mm. (d) Cell fragments in the lens cavity of the framed area in (c). Cell fragments (white arrows) are generally round, 1.8e2.3 mm in diameter. Lens epithelial cell of the lens vesicle (LE); lens cavity (LC), scale bar ¼ 1 mm.
3.1. Localization of F4/80 positive cells in the lens primordium At around 10 days of gestation, the optic vesicle began to invaginate to form the optic cup, and, at the same time, the lens placode became infolded to form the lens pit. A few positive cells appeared not only in the lumen of the pit but also in the surface ectoderm at the periphery of the lens placode (Fig. 1a). F4/80 positive cells then increased in number, and could be found in the lens stalk, adjacent mesenchymal tissues and the narrow space between the lens vesicle and the optic cup (Fig. 1b). The lens vesicle became detached from the surface
ectoderm at day 11.5, and F4/80 positive cells could be recognized in both the anterior epithelial wall and the lens cavity (Fig. 1c). However, hardly any positive cells at all could be detected in the posterior wall of the lens vesicle (Fig. 1d). During primary lens fiber differentiation around day 13, the narrowing lens cavity contained numerous positive cells. Most of the positive cells in the cavity attached to the anterior epithelial wall of the lens vesicle, but a few positive cells were also found within the lens epithelial cell layer. The thickening posterior wall of the vesicle, on the contrary, contained no positive cells (Fig. 1e). Since positive cells in the space in front of the lens vesicle were also attached to the outer lens surface, the
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Fig. 3. Electron micrographs of the lens vesicle at 11.5 days of gestation. (a) A low-powered micrograph of the lens vesicle. The lens stalk (LS) can be recognized between the surface ectoderm (EC) and the lens vesicle (LV). Macrophages (*) are located in the ectoderm epithelium, lens stalk and lens cavity; scale bar ¼ 10 mm. (b) A mitotic macrophage in the lens cavity of (a). This macrophage has numerous villous projections (arrows) from the cell surface and includes several phagosomes (P) derived from a dying lens epithelial cell; scale bar ¼ 2 mm. (c) A macrophage in the lens epithelium of (a). This macrophage (Mp) includes a large phagosome derived from an epithelial cell having a condensed nucleus (arrow); scale bar ¼ 2 mm. (d) A detached epithelial cell in the lens cavity. A detached epithelial cell (DE) in lens cavity (LC) retains a junctional association (arrow) with an anterior lens wall cell; scale bar ¼ 2 mm.
epithelial layer appeared to be sandwiched by numerous macrophages (Fig. 1e). After the lens cavity was filled, positive cells disappeared, and thereafter the lens did not contain any F4/80 positive cells at all. Positive cells were observed not only in the mesenchyme between the cornea and the lens
but also in the primitive vitreous cavity between the lens and the prospective retina (Fig. 1f). The number of F4/80 positive cells in the primary vitreous cavity behind the lens appeared to be markedly increased after 14 days of gestation (Fig. 1d, e).
Fig. 4. Electron micrographs of the lens vesicle at 13.5 days of gestation. (a) A low-powered micrograph of the lens cavity. Arrows indicate macrophages in the lens cavity (LC). Lens epithelium (LE), scale bar ¼ 10 mm. (b) A macrophage in the lens cavity of (a). This macrophage has numerous projections on the cell surface and includes numerous vacuoles (*) in the cytoplasm. The arrow indicates cell contacts between the macrophage and epithelial cells of the anterior wall of the lens vesicle; scale bar ¼ 2 mm.
K. Nishitani, K. Sasaki / Experimental Eye Research 83 (2006) 223e228
3.2. Ultrastructure of macrophages in the lens primordium At 10.5 days of gestation, the lens placode become indented, and then the lens pit was formed. In the epithelium at the margin of the lens pit, numerous dying cells appeared, and the dead cells were engulfed in the epithelium by neighboring epithelial cells and scavenger macrophages (Fig. 2a). Some epithelial cells appeared to release small cytoplasmic fragments into the lens pit (Fig. 2b). At the 11.5 days of gestation, the lens pit was closed to form the lens cavity where many small cell fragments could be observed. The fragments were characteristically located near the anterior wall of the lens vesicle (Fig. 2c, d). The lens cavity contained two different kinds of cellular elements; scavenger macrophages and fragments from epithelial cells (Fig. 3). At 12 days of gestation, when the lens stalk disappeared, the round lens vesicle separated from the surface ectoderm. Both the lens vesicle and the mesenchyme between the surface ectoderm and the lens vesicle contained numerous macrophages. The cytoplasm held phagosomes derived from dying epithelial cell debris and cell fragments (Fig. 3a). Macrophages in mitosis were often distributed in the lens cavity (Fig. 3b). Macrophages in the lens epithelium contained the dying epithelial cells with nuclei composed of highly condensed chromatin (Fig. 3c), a typical sign of apoptosis. The lens cavity also sometimes contained epithelial cells which had detached of the lens anterior wall (Fig. 3d). At 13.5 days of gestation, macrophages were often seen making intimate contact with the posterior surface of the lens epithelium in the cavity (Fig. 4a). The macrophages had very large vacuoles in their cytoplasm, and often attached to the surface of lens epithelial cells, and were characterized by fingerlike projections (Fig. 4b).
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origins; scavenger macrophages, dying cells and cell components derived from epithelial cells. In the closed space, not neighboring epithelial cells but scavenger macrophages could be responsible for the removal of cellular elements which fall out of the epithelium. Our observation also showed that macrophages displayed a characteristic localization in the space of the lens cavity. Studies on lens development have demonstrated that the lens cavity in the lens vesicle is fated to disappear due to elongation of the primary lens fiber (Davidson, 1981). During the closing process, epithelial cells undergoing apoptosis were frequently observed in the epithelial stalk, adjacent ectoderm and anterior wall of the lens vesicle (Mohamed and Amemiya, 2003). As shown in our results, apoptotic cells, showing chromatin agglutination, were present in the lens epithelium but not in the primary lens fiber at this stage. At the same time, the epithelium began to be sandwiched by numerous F4/80 positive cells, although positive cells could not be found at all in the posterior wall consisting of primary lens fiber. During the process of apoptosis in mammalian systems, functional detail concerning the phagocytosis of apoptotic cells by macrophages was elucidated (Potten and Wilson, 2004). Macrophages are known to recognize specific changes in dying cells, and engulf them. Then the macrophages are stimulated to produce a signaling molecule of transforming growth factor-b1. The most important functional significance of lens macrophages might be as a scavenger for the removal of either apoptotic cells or cell fragments. The characteristic localization of macrophages in developing lens could be considered to reflect the occurrence of apoptosis in the developing lens. The physiological significance of cytoplasmic fragments of epithelial cells in the lens cavity should possibly be considered with regard to the developmental change in the lens epithelial cells.
4. Discussion
Acknowledgements
The present study clearly showed that scavenger macrophages are intimately involved in early lens formation. As mentioned above, numerous F4/80 positive cells were observed in the ectoderm, lens vesicle, lens cavity and surrounding mesenchymal tissue from 10.5 days to 12 days of gestation. These F4/80 positive cells could be identified as macrophages by their ultrastructural features, and they often held large phagosomes probably derived from either dying epithelial cells or cell fragments. At the early stage of lens formation, cell death by apoptosis has previously been observed in the lens primordium (Wride, 1996; Laemle et al., 1999; Mohamed and Amemiya, 2003). Our results showed that, in addition to the neighboring epithelial cells, macrophages appeared in the epithelium at the border of the lens placode, and removed these dead cells. Pei and Rhodin (1970) reported that, within the pit and cavity of the lens vesicle, degenerating cells could be found, and, in addition to the dying cells, cytoplasmic fragments of epithelial cells also appeared to fall into either the lens pit or the lens cavity. In the cavity of the lens vesicle, therefore, there exist several cellular elements from multiple
The authors wish to thank Mr. K. Uehira, Mr. T. Suda and Mrs. M. Suda for their skillful technical assistance. This work was supported in part by a research project grant from Kawasaki Medical School (140-201,2002).
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