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cup during early eye morphogenesis in the rat
Exp. Eye Res. (1981) 33~ 447458
The Spatial Relationship Between Presumptive Lens and Optic Vesicle/Cup During Early Eye Morphogenesis in the Rat J. W. M c A v o r
Department Histology and Embryology, The University of Sydney , Sydney 2006, New South Wales, Australia (Received 14 November 1980 and accepted 3 March 1981, London) Mesodermal cells are pushed aside as the optic vesicle approaches the ectoderm but some remain sandwiched between the two tissues. Some of these sandwiched cells degenerate, others persist at least until the lens pit stage. During the period of close association between the optic vesicle/cup and presumptive lens there are cytoplasmic processes extending from both tissues. The processes are detected early on the 12th day before the lens placode forms. Processes are abundant between lens placode and optic vesicle. They are also detected between lens pit and optic cup but are not as common as between placode and vesicle. Cytoplasmic processes are not detected after 13 days when the space between the tissues has widened to form primary vitreous. The processes may be necessary for adequate communication between the tissues during the early period of their interaction. Coated pits and vesicles are commonly found in the processes. It is generally accepted that coated pits and vesicles are involved in receptor-mediated endocytosis~ therefore it is suggested that they may be involved in the uptake of inducer molecules from the optic vesicle/cup. Coated pits and vesicles are also abundant at 13 days in the basal cytoplasm of the cells in the posterior part of the lens vesicle which form the primary fibre cells. In addition to the cytoplasmic processes there is a network of fibrils in the interspace. These are associated with the cytoplasmic processes and the basal surfaces of the presumptive lens and optic vesicle/cup. These are probably important for the co-ordinated invagination of lens placode and optic vesicle to form tens pit and optic cup, respectively. The vitreous arises from the space between presumptive lens and optic vesicle. There is a noticeable build-up of basal lamina material as development proceeds. Some of this extends into the interspace and is intimately associated with clumps of amorphous material. Thus at least some of the earliest vitreous constitutents are laid down by the epithelial cells !ining the interspace. Key words: lens; morphogenesis; optic vesicle; cell processes; rats; embryonic induction,
1. I n t r o d u c t i o n I n v e r t e b r a t e s t h e r e is a close spatial relationship b et w een p r e s u m p t i v e lens an d optic v e s i c l e / c u p during eye morphogenesis. I t has been d e m o n s t r a t e d e x p e r i m e n t a l l y t h a t the p r o x i m i t y of th e optic vesicle has a causal influence on lens differentiation. F o r example, in a m p h i b i a n s (Lewis, 1907) and chicks (Alexander, 1937) t r a n s p l a n t a t i o n of the optic vesicle to th e t r u n k results in a lens f o r m i n g from t r u n k ectoderm. T h u s lens m o rp h o g en es is in v i v o is influenced in some w a y by the presence of optic vesicle. There are t wo s e p a r a t e ways t h a t the optic vesicle m a y e x e r t its lens inducing influence: (1) b y displacing m e s o d e r m a l cells as it a p p r o a c h e s the ectoderm. I t has been clearly shown t h a t m e s o d e r m has an i n d u c t i v e influence on ep i d er m al different i at i o n (see for e x a m p l e Sengel, 1958} and (2) b y p r o d u c i n g an i n d u c t i v e signal t h a t p r o m o t e s lens m o r p h o g e n e s i s from e c t o d e r m . A t h i r d possibility is a c o m b i n a t i o n of these two, i.e. t h e optic vesicle largely displaces m e s o d e r m which p r o v i d e s cues for skin differentiation with optic vesicle which provides cues for lens differentiation. Therefore, a basic r e q u i r e m e n t for u n d e r s t a n d i n g t h e m e c h a n i s m of lens i n d u c t i o n is 9 1981 Academic Press Inc. (London) Limited
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to describe the spatial relationships between presumptive lens ectoderm, mesoderm and optic vesicle. In recent years the chick has received most attention. I t has been shown that at their closest period of association a space (interspace) remains between optic vesicle and presumptive lens ectoderm that measures 1-3 #m (Hunt, 1961) and that all mesodermal cells are excluded from the interspace (Wakely, 1977). Comprehensive light and electron microscope studies (Hunt, 1961 ; Weiss and Jackson, 1961; Silver and Wakely, 1974; Hendrix and Zwaan, 1975) did not report any intercellular communication between optic vesicle and presumptive lens ectoderm in chicks. Similar observations were made by Cohen (1961) in an ultrastructural study in mice. However, Mann (1950) in a light microscope study of humans described cone shaped processes bridging the interspace between the two tissues at least up to the lens pit stage. Cytoplasmic processes were also reported in a preliminary transmission electron microscope study and in some cases they were shown to traverse the interspace and make contact with theopposing tissue (McAvoy, 1980). This present communication extends this work and includes an analysis of the interspace by scanning electron microscopy.
2. M a t e r i a l s a n d M e t h o d s Albino Wistar rats were mated in the evening and separated the next morning and this was counted day 0 of pregnancy, Females were weighed every 2 days and those that showed at least a 10 % weight increase by 11 days were judged pregnant. Pregnant rats were killed and the embryos fixed in Karnovsky's for 1 hr (Karnovsky, 1965), post fixed in 1% osmium tetroxide for 1 hr and stained with 2 % aqueous uranyl acetate for 30 rain. The embryos were dehydrated in ethanol and embedded in Spurr's mixture (Spurr, 1969). Sections were cut at 1 #m and 60-90 nm. Micron sections were stained with toluidine blue. The ultrathin sections were stained with uranyl acetate and lead citrate and viewed with a Jeol 100CX electron microscope. For scanning electron microscopy the embryos were fixed in Karnovsky's for 4 or 24 hr then placed in buffer. In each embryo a meridional section was cut through the centre of the eye primordium using a razor blade. The different fixation times did not appreciably affect the sectioning properties of the embryos. The embryos were post fixed in 1% osmium tetroxide for 1 hr, dehydrated in ethanol and critical-point dried. The specimens were gold coated and examined in an ISI IIIA scanning electron microscope.
3. R e s u l t s In rats the optic vesicles become closely associated with ectoderm at I i days of embryonic development. Mesodermal cells which underlie the ectoderm are pushed aside but some remain sandwiched between the two tissues (Fig. 1). Some of these remaining cells show signs of degeneration as evidenced by nuclear pycnosis (Figs 1 and 2). It is not clear if all the mesodermal cells degenerate or if any contribute to the cells and vessels of the primary vitreous that form in the space between the tissues at 13 days (see later). In the latter part of the l l t h day the ectoderm thickens to form the lens placode and begins invagination to form the lens pit (Fig. 3). During the period of close association between the presumptive lens and optic vesicle there is a space between these tissues known as 'the interspace' (Silver and Wakely, 1974). In rats t h e interspace does not vary much in width during the 11th day and averages 5"7 + 0"9 #m (S.D.). Cytoplasmic processes are prominent in the interspace during the 11th day (Fig. 3). The processes are extensions of the basal part of the cells. There appear to be two main types of processes; thick and thin processes. At their origin
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FIG. 1. Optic vesicle (OV) and presumptive lens ectoderm (PLE) at 11 days of development. Mesodermal cellsare sandwichedbetween the two tissues. One of the mesodermalceilsis pycno~ic(arrow). x 250. FIG. 2. Pycnotic cell in the space between optic vesicle and presumptive lens. The nuclear membrane has partially broken down. x 24500. the thick processes generally have a diameter similar to that of the cell but taper as they extend into the interspace (Fig. 4). Thin processes sometimes extend from the thick processes [Figs 4 (b) and 5 (b), (c)] or m a y extend directly from the bases of the cells [Fig. 5 (a)]. In some cases these processes bridge the interspace and make contact with the opposing tissue [Figs 4(b) and 5 (a), (b), (c)]. In the transmission electron microscope the thick processes are seen to contain large numbers of vesicles, some of which have an electron dense coat on their membrane (Fig. 6). These vesicles probably arise by micropinocytosis because similarly coated pits are also commonly seen in the plasma membrane of the processes. Thin processes that extend from thick processes are largely comprised of fine filaments and contain few, if any, cytoplasmic organelles [Fig. 6(b); see also McAvoy (1980b)]. In addition to the cytoplasmic processes there is a network of fibrils "in the interspace. These are associated with the cytoplasmic processes and the basal surfaces of the presumptive lens and optic vesicle (Figs 4 and 5). Some of these fibrils have a periodicity characteristic of collagen fibrils (McAvoy, 1980). By 12 days of embryonic development the lens placode and optic vesicle have invaginated to form the lens pit and optic cup, respectively (Fig. 7). The interspace averages 5"7+1"6#m so there is essentially no change in width from 11 days. Cytoplasmic processes are not as common as at 11 days but they can still be detected [Fig. 8 (b)]. Mesodermal cells are common in the interspace particularly in the vicinity 16
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FIG. 3. Late in the l l t h day the lens placode (LP) and optic vesicle (OV) begin invagination. Cytoplasmic processes extend across the space between tissues (arrow). A mesodermal cell remains sandwiched between the tissues (open arrow), x 800. Fro. 4 (a), (b). Thick cytoplasmic processes extend from the lens plaeode (LP) and optic vesicle (OV). Fibrils are often associated with the thick processes. In (b) a thin process branches from a thick process and appears to make contact with the optic vesicle. (a) x 4500; (b) x 5000.
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of the foetal fissure. Cells from outside the developing eye have access to the interspace via the foetal fissure and presumably most of the mesodermal cells of the vitreous enter through this passage. Mesodermal cells t h a t are trapped in the interspace between the optic vesicle and presumptive lens m a y also contribute to the cell population of the vitreous since they are present at all stages of early development. However these are few in number and it is not clear if any remain viable.
Fic. 5. (a) Thin processesextend across the space between the presumptive lens (PL) and optic vesicle (OV). The processes in (b) and (c) appear to extend from thick processes (arrows) Fibrils are associated with the processes as well as the basal regions of the epithelial cells. • 6000. The main feature'of the interspace at 12 days is the dense meshwork of fibrils (Fig. 8). These are associated with the mesodermal cells as well as the two cellular layers. In the transmission electron microscope clumps of amorphous material are also detected at this stage. This material is present in the interspace but m a y also be associated with the basal lamina of the presumptive lens and optic cup [Fig. 9 (a), (b)]. The basal laminae of these two tissues is highly elaborated in some regions. This is particularly obvious in the case of the optic cup where it often forms several irregularly arranged layers and extends into the interspace. Thus at least some of the earliest vitreous constituents are laid down by the epithelial ceils lining the interspace. This has also been established in chicks b y Silver and Wakely (1974). By 13 days the lens vesicle has formed and cells in the posterior of the vesicle have commenced to elongate to form the primary lens fibres. The interspace is substantially wider than at 12 days and blood vessels are present often containing blood cells at various stages of maturation (Fig. 10). The endothelial cells of the vessels are said to arise from the mesodermal cells t h a t migrate into the interspace via the foetal fissure (Mann, 1950). The basal laminae of the lens and optic cup are obviously elaborated at this stage. In the lens there are 5-6 layers of basal laminae and together these form the incipient capsule [Fig. 11 (a)]. In the optic cup the basal laminae are not present in regularly arranged layers but, as at 12 days, extend into the interspace [Fig. 11 (b)]. 16-2
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Fio. 6. (a) A thick cytoplasmic process extends across the interspace (IS) from the presumptive lens (PL) to the optic vesicle (OV). (b) The thick cytoplasmic process has a thin process branching from it. Another process closely associated with the thick process (arrow) contains numerous fine filaments. This process may have been attached to the basal lamina of the optic vesicle but appears to have broken aw%v leaving part of it still attached (open arrow). The thick process contains many vesicles some of which are coated vesicles. A coated pit is indicated by the horizontal arrow. (a) x 3500; (b) x 11400. T h e b a s a l c y t o p l a s m of t h e lens cells c o n t a i n s n u m e r o u s c o a t e d pits a n d vesicles [Fig. 11 (a)] s i m i l a r to t h o s e p r o m i n e n t l y f o u n d at 11 d a y s in t h e c y t o p l a s m i c processes. T h e s e are also p r e s e n t in t h e o p t i c cup cells b u t are n o t so a b u n d a n t . E l e c t r o n dense bodies t h a t are a s s o c i a t e d w i t h t h e c o a t e d vesicles are p r o b a b l y lysosomes.
4. D i s c u s s i o n T h e s p a t i a l r e l a t i o n s h i p b e t w e e n o p t i c vesicle, p r e s u m p t i v e lens e c t o d e r m a n d m e s o d e r m was i n v e s t i g a t e d d u r i n g t h e e a r l y s t a g e s of e y e m o r p h o g e n e s i s . M e s o d e r m a l cells are p u s h e d aside as t h e o p t i c vesicle a p p r o a c h e s t h e e c t o d e r m b u t s o m e r e m a i n s a n d w i c h e d b e t w e e n t h e t w o tissues. S o m e of t h e s e o b v i o u s l y d e g e n e r a t e b u t o t h e r s
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FIG. 7. Lens pit (LP) and optic cup (OC) at 12 days. Mesodermal cells are present in the space between these tissues particularly in the vicinity of the foetal fissure (F). • 360. FIG. 8. (a) The interspace at 12 days is filled by a network of fibrils. These connect up with both the mesodermal cells and the epithelial cells. • 2100. (b) Thick (T) and thin (t) cytoplasmic processes are sometimes present. • 2700.
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FIG. 9. The interspace at 12 days contains mesodermal cells and clumps of amorphous material. (a) The basal lamina, particularly on the optic vesicle (OV) side sometimes extends into the interspace (arrow) and (b) forms several layers drawn out into many folds. Amorphous material is often associated with the basal lamina (arrow) (a) x 2800; (b) x l1550. persist until the lens p i t stage. A t this stage m e s o d e r m a l cells from outside the optic p r i m o r d i u m have access to a n d are t h o u g h t to enter, the interspace v i a the foetal fissure (Mann, 1950). I t is n o t possible in this morphological s t u d y to d e t e r m i n e whether all of the sandwiched m e s o d e r m a l cells degenerate, or if a n y of t h e m r e m a i n viable a n d c o n t r i b u t e to t h e composition of the vitreous. However, it is c o m m o n l y assumed in h u m a n s t h a t the vascular elements of the vitreous are formed t o t a l l y from m i g r a t o r y m e s o d e r m a l cells (see Mann, 1950). I t has been suggested from studies on an a n o p h t h a l m i e strain of mice t h a t m e s o d e r m a l cells have an i n h i b i t o r y effect on lens differentiation (Silver and Hughes, 1974). I n these mice viable m e s o d e r m a l cells r e m a i n between the p r e s u m p t i v e lens and optic vesicle a n d are sometimes seen in mitosis, whereas in n o r m a l mice m e s o d e r m becomes necrotic and is resorbed. I n the r a t it is not clear if all the m e s o d e r m a l cells become necrotic during the period of close association of the tissues, i.e. 12th a n d 13th
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FIG. 10. The interspace at 13 days contains many mesodermal cells. Blood vessels have formed and contain blood cells at various stages of maturation, x 2300. Fro. 11. (a) The lens capsule consists of approximately 6-7 layers of basal laminae. Coated pits (arrow) and vesicles are common along with lysosomc:like structures (L). x 28000. (b) The basal laminae of the optic cup (OC) forms several irregular layers. • 10000.
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days of development. Nevertheless viable mesodermal cells, if any, are few in number and obviously do not interfere with the interaction between presumptive lens and optic vesicle/cup. This study shows that during the period of close association between the optic vesicle/cup and presumptive lens there are cytoplasmic processes extending from both tissues. The processes are detected early on the 12th day before the lens placode forms. Processes are also abundant between lens placode and optic vesicle. They are not as common, although still detected between lens pit and optic cup. The processes are not detected after 13 days when the space between the two tissues has widened to form the primary vitreous. The role of the processes is not clear although it is tempting to speculate that they may be important for the transmission of inducing signals between the tissues. Saxen (1977) has proposed that transmission of information falls into two categories; long range and short range. The former is mediated by substances that can diffuse over large distances, whereas the latter depends on cellular contact with the inducer whether it is with an inducing matrix or cells of another type. In the case of the lens some of the cytoplasmic processes make contact with the opposing tissue (see also McAvoy, 1980a). Therefore, on this basis this interaction would be classified as an example of cell mediated induction. However, other experiments and observations do not, in general, support this view. Karkinen-Ji~skel~inen (1978) showed by in vitro transfilter experiments that lens induction took place in chicks even though no cellular contacts were detected by electron microscopy in the filter interposed between ectoderm and optic vesicle. Cellular contacts have not been detected in chicks in vivo despite comprehensive light and electron microscope studies (Hunt, 1961; Weiss and Jackson, 1961; Silver and Wakely, 1974; Hendrix and Zwaan, 1975.) Thus experimental evidence indicates that lens induction can take place in chicks without cell contacts between presumptive lens and optic vesicle. Consistent with this are the m a n y observations that there are no cell processes between these tissues in vivo. On the other hand cytoplasmic processes have been detected in humans (Mann, 1950) and now in rats. Thus the situation with regard to these processes in vertebrates is not clear. They may be present in some species but not all. One difference between chicks and rats is the width of the interspace. In chicks the interspace ranges from 1-3 #m (Hunt, 1961) whereas in rats it averages 5'7 +0"9 #m. Thus in rats and perhaps in other mammals it could be argued t h a t the processes effectively narrow the space between the tissues. In this case cell contact may not be an important event but the processes may be necessary for adequate communication between presumptive lens and optic vesicle/cup during the early period of their interaction. Complete lens differentiation depends on a continuous interaction with the optic cup (see, for example, Le Cron, 1907 ; Muthukkaruppan, 1965) as does the differentiation of epithelial cells into fibres during lens growth (Coulombre and Coulombre, 1963; Muthukkaruppan, 1965; Yamamoto, 1976). This inducer must be a diffusable substance since it can be extracted from the vitreous (Beebe, Feagans and Jebens, 1980) and is present in retina conditioned cell free medium (McAvoy, unpublished results). Consistent with this is the observation that no cytoplasmic processes are present at 14 days when the primary fibres are forming. However this does not rule out the need for cell contact at 11 and 12 days since the inductive signal for fibre formation need not be the same as the signal for the initiation of lens morphogensis (See McAvoy, 1980).
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Another role of the processes m a y be purely mechanical. The cytoplasmic processes together with the fibrils that extend from them and from the basal laminae of the presumptive lens and optic vesicle/cup m a y be important for the adherence of these two tissues. Zwaan and Hendrix (1973) suggested that this adhesion is a prerequisite for invagination. In the chick they showed t h a t the area of contact between presumptive lens and optic vesicle becomes fixed soon after these tissues become associated and suggested that the increased population pressure caused by cell proliferation results in lens cell elongation and finally invagination. Recently Schook (1980) has stressed the importance for invagination of an active extrusion of apical cell cytoplasm in cells of the lens placode. He suggests that because of this extrusion the placode cells which are held together at the apical side, gradually shrink at their apex and grow out in a basal direction. In any case adhesion between presumptive lens and optic vesicle is obviously important in the co-ordination of invagination of lens placode and optic vesicle to form lens pit and optic cup, respectively. A potentially interesting observation is that the thick processes contain an abundance of micropinocytotic pits and vesicles, m a n y of them similar to the coated pits and coated vesicles described by Goldstein, Anderson and Brown (1979). The pits and vesicles are also present in the basal cytoplasm of the lens and optic vesicle cells but are much more abundant in the processes. Coated pits and vesicles have been shown to be involved in receptor-mediated endocytosis, i.e. a process whereby selected extracellular proteins or peptides are first bound to specific cell surface receptors which are clustered in specialized regions of the cell surface and form coated pits. These are internalized to form intracellular coated vesicles. I t is now generally recognized t h a t receptor-mediated endocytosis has a fundamental role in growth, nutrition and differentiation of cells. Consequently during embryonic development it m a y be envisaged t h a t coated pits and vesicles play an essential role, at least in some forms of tissue interactions, in uptake of molecules from associated tissues which m a y determine the fate of the receptive cells. The presence of coated pits and vesicles indicate that the presumptive lens cells are taking up specific environmental signals. This coincides with the initiation of lens morphogenesis and it is tempting to speculate that the presumptive lens cells with the aid of cytoplasmic processes take up an inducer substance from the optic vesicle by receptor-mediated endocytosis. Coated pits and vesicles are also abundant at 13 days in the basal cytoplasm of the cells in the posterior part of the lens vesicle which form the primary fibre cells. I t could be again argued t h a t this represents the uptake of specific molecules involved in the initiation of fibre differentiation, although these are not necessarily the same molecules t h a t trigger lens morphogenesis. Thus it could be concluded that lens induction depends on the displacement of mesodermal cells, which provide cues for skin differentiation, by the optic vesicle which provides cues for lens morphogenesis. The lens cells m a y take up the inducing molecules by receptor-mediated endocytosis particularly in cytoplasmic processes which narrow the space between the interacting tissues. ACKNOWLEDGMENTS I am grateful to Melody Abbott for her technical assistance and to the University of Sydney Electron Miscroscope Unit for assisting me in the use of their Scanning Electron Miscroscope facility. Support for this research was provided by grant no. 1 RO1 Ey 03177 awarded by the N.E.I. Department of Health, Education and Welfare, U.S.A.
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Alexander, L.E. (1937). An experimental study of the role of optic cup and overlying ectoderm in lens formation in the chick embryo. J. Exp. Zool. 75, 41-73. Beebe, D. C., Feagans, D. E. and Jebens, H. A. H. (1980). Lentropin: a factor in vitreous humor which promotes lens fiber cell differentiation. Proc. Nat. Acad. Sci. U.S.A. 77, 490-3. Cohen, A. I. (1961). Electron microscopic observations of the developing mouse eye. I. Basement membranes during early development and lens formation. Dev. Biol. 3. 297-316. Coulombre, J. L. and Coulombre, A. J. (1963). Lens development: fiber elongation and lens orientation. Science 142, 1489. Goldstein, J. L., Anderson, R. G. W. and Brown, M. S. (1979). Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature (London) 279, 679-85. Hendrix, R.W. and Zwaan, J. (1975). The matrix of the optic vesicle presumptive lens interface during induction of the lens in the chicken embryo. J. Embryol. Exp. Morph. 33, 1023-49. Hunt, H. H. (1961). A study of the fine structure of the optic vesicle and lens placode of the chick embryo during induction. Dev. Biol. 3, 175-209. Karkinen-J~gskel~inen, M. (1978). Transfilter lens induction in avian embryo. Differentiation 12, 31-7. Karnovsky, M. J. (1965). A formaldehyde-gluteraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137a. Le Cron, W.L. (1907). Experiments on the origin and differentiation of the lens in Amblystoma. Am. J. Anat. 6, 245-57. Lewis, W. H. (1907). Lens formation from strange ectoderm in Rana sylvatica. Am. J. Anat. 7, 145-69. Mann, I. (1950). The Development of the Human Eye. Grune & Stratton, New York. McAvoy, J. W. (1980a): Induction of the eye lens. Differentiation 17, 137-49. McAvoy, J. W. (1980b). Cytoplasmic processes interconnect lens placode and optic vesicle during eye morphogenesis. Exp. Eye Res. 31,527-34. Muthukkaruppan, V. (1965). Inductive tissue interaction in the development of the mouse lens in vitro. J. Exp. Zool. 159, 269-87. Saxen, L. (1977). Morphogenetic tissue interactions: an introduction. In Cell Interaction in Differentiation (Ed. M. Karkinen-Jgs L. Saxen and L. Weiss). Pp. 145-51. Academic Press, London. Schook, P. (1980). Morphogenetic movements during the early development of the chick eye. An ultrastructural and spatial reconstructive study. A. Invagination of the lens placode. Acta Morphol. Neerl. Scand. lg, 133--57. Sengel, P. (1958). gecherches exp6rimentales sur la diff~renciation des germes plumaires et du pigment de la peau de l'embryon du poulet en culture in vitro. Ann. Sci. Nat. Zool. 20, 431-514. Silver, J. and Hughes, A. F. W. (1974). The relationship between morphogenetic cell death and the development of congenital anophthalmia. J. Comp. Near. 157, 281-302. Silver, P. H. S. and Wakely, J. (1974). Fine structure, origin and fate of extracellular materials in the interspace between the presumptive lens and presumptive retina of the chick embryo. J. Anat. 118, 19-31. Spurr, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43. Wakely, J. (1977). Scanning electron microscope study of the extracellular matrix between presumptive lens and presumptive retina of the chick embryo. Anat. Embryol. 150, 163-70. Weiss, P. and Jackson, S. F. (1961). Fine-structural changes associated with lens determination in the avian embryo. Dev. Biol. 3, 532-54. Yamamoto, Y. (1976). Growth of lens and ocular environment: role of neural retina in the growth of mouse lens as revealed by an implantation experiment. Development, Growth and Differentiation lg, 273-78. Zwaan, J. and Hendrix, R.W. (1973). Changes in cell and organ shape during early development of the ocular lens. Am. Zool. 13, 1039-49.
The Spatial Relationship Between Presumptive Lens and Optic Vesicle/Cup During Early Eye Morphogenesis in the Rat J. W. M c A v o r
Department Histology and Embryology, The University of Sydney , Sydney 2006, New South Wales, Australia (Received 14 November 1980 and accepted 3 March 1981, London) Mesodermal cells are pushed aside as the optic vesicle approaches the ectoderm but some remain sandwiched between the two tissues. Some of these sandwiched cells degenerate, others persist at least until the lens pit stage. During the period of close association between the optic vesicle/cup and presumptive lens there are cytoplasmic processes extending from both tissues. The processes are detected early on the 12th day before the lens placode forms. Processes are abundant between lens placode and optic vesicle. They are also detected between lens pit and optic cup but are not as common as between placode and vesicle. Cytoplasmic processes are not detected after 13 days when the space between the tissues has widened to form primary vitreous. The processes may be necessary for adequate communication between the tissues during the early period of their interaction. Coated pits and vesicles are commonly found in the processes. It is generally accepted that coated pits and vesicles are involved in receptor-mediated endocytosis~ therefore it is suggested that they may be involved in the uptake of inducer molecules from the optic vesicle/cup. Coated pits and vesicles are also abundant at 13 days in the basal cytoplasm of the cells in the posterior part of the lens vesicle which form the primary fibre cells. In addition to the cytoplasmic processes there is a network of fibrils in the interspace. These are associated with the cytoplasmic processes and the basal surfaces of the presumptive lens and optic vesicle/cup. These are probably important for the co-ordinated invagination of lens placode and optic vesicle to form tens pit and optic cup, respectively. The vitreous arises from the space between presumptive lens and optic vesicle. There is a noticeable build-up of basal lamina material as development proceeds. Some of this extends into the interspace and is intimately associated with clumps of amorphous material. Thus at least some of the earliest vitreous constitutents are laid down by the epithelial cells !ining the interspace. Key words: lens; morphogenesis; optic vesicle; cell processes; rats; embryonic induction,
1. I n t r o d u c t i o n I n v e r t e b r a t e s t h e r e is a close spatial relationship b et w een p r e s u m p t i v e lens an d optic v e s i c l e / c u p during eye morphogenesis. I t has been d e m o n s t r a t e d e x p e r i m e n t a l l y t h a t the p r o x i m i t y of th e optic vesicle has a causal influence on lens differentiation. F o r example, in a m p h i b i a n s (Lewis, 1907) and chicks (Alexander, 1937) t r a n s p l a n t a t i o n of the optic vesicle to th e t r u n k results in a lens f o r m i n g from t r u n k ectoderm. T h u s lens m o rp h o g en es is in v i v o is influenced in some w a y by the presence of optic vesicle. There are t wo s e p a r a t e ways t h a t the optic vesicle m a y e x e r t its lens inducing influence: (1) b y displacing m e s o d e r m a l cells as it a p p r o a c h e s the ectoderm. I t has been clearly shown t h a t m e s o d e r m has an i n d u c t i v e influence on ep i d er m al different i at i o n (see for e x a m p l e Sengel, 1958} and (2) b y p r o d u c i n g an i n d u c t i v e signal t h a t p r o m o t e s lens m o r p h o g e n e s i s from e c t o d e r m . A t h i r d possibility is a c o m b i n a t i o n of these two, i.e. t h e optic vesicle largely displaces m e s o d e r m which p r o v i d e s cues for skin differentiation with optic vesicle which provides cues for lens differentiation. Therefore, a basic r e q u i r e m e n t for u n d e r s t a n d i n g t h e m e c h a n i s m of lens i n d u c t i o n is 9 1981 Academic Press Inc. (London) Limited
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to describe the spatial relationships between presumptive lens ectoderm, mesoderm and optic vesicle. In recent years the chick has received most attention. I t has been shown that at their closest period of association a space (interspace) remains between optic vesicle and presumptive lens ectoderm that measures 1-3 #m (Hunt, 1961) and that all mesodermal cells are excluded from the interspace (Wakely, 1977). Comprehensive light and electron microscope studies (Hunt, 1961 ; Weiss and Jackson, 1961; Silver and Wakely, 1974; Hendrix and Zwaan, 1975) did not report any intercellular communication between optic vesicle and presumptive lens ectoderm in chicks. Similar observations were made by Cohen (1961) in an ultrastructural study in mice. However, Mann (1950) in a light microscope study of humans described cone shaped processes bridging the interspace between the two tissues at least up to the lens pit stage. Cytoplasmic processes were also reported in a preliminary transmission electron microscope study and in some cases they were shown to traverse the interspace and make contact with theopposing tissue (McAvoy, 1980). This present communication extends this work and includes an analysis of the interspace by scanning electron microscopy.
2. M a t e r i a l s a n d M e t h o d s Albino Wistar rats were mated in the evening and separated the next morning and this was counted day 0 of pregnancy, Females were weighed every 2 days and those that showed at least a 10 % weight increase by 11 days were judged pregnant. Pregnant rats were killed and the embryos fixed in Karnovsky's for 1 hr (Karnovsky, 1965), post fixed in 1% osmium tetroxide for 1 hr and stained with 2 % aqueous uranyl acetate for 30 rain. The embryos were dehydrated in ethanol and embedded in Spurr's mixture (Spurr, 1969). Sections were cut at 1 #m and 60-90 nm. Micron sections were stained with toluidine blue. The ultrathin sections were stained with uranyl acetate and lead citrate and viewed with a Jeol 100CX electron microscope. For scanning electron microscopy the embryos were fixed in Karnovsky's for 4 or 24 hr then placed in buffer. In each embryo a meridional section was cut through the centre of the eye primordium using a razor blade. The different fixation times did not appreciably affect the sectioning properties of the embryos. The embryos were post fixed in 1% osmium tetroxide for 1 hr, dehydrated in ethanol and critical-point dried. The specimens were gold coated and examined in an ISI IIIA scanning electron microscope.
3. R e s u l t s In rats the optic vesicles become closely associated with ectoderm at I i days of embryonic development. Mesodermal cells which underlie the ectoderm are pushed aside but some remain sandwiched between the two tissues (Fig. 1). Some of these remaining cells show signs of degeneration as evidenced by nuclear pycnosis (Figs 1 and 2). It is not clear if all the mesodermal cells degenerate or if any contribute to the cells and vessels of the primary vitreous that form in the space between the tissues at 13 days (see later). In the latter part of the l l t h day the ectoderm thickens to form the lens placode and begins invagination to form the lens pit (Fig. 3). During the period of close association between the presumptive lens and optic vesicle there is a space between these tissues known as 'the interspace' (Silver and Wakely, 1974). In rats t h e interspace does not vary much in width during the 11th day and averages 5"7 + 0"9 #m (S.D.). Cytoplasmic processes are prominent in the interspace during the 11th day (Fig. 3). The processes are extensions of the basal part of the cells. There appear to be two main types of processes; thick and thin processes. At their origin
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FIG. 1. Optic vesicle (OV) and presumptive lens ectoderm (PLE) at 11 days of development. Mesodermal cellsare sandwichedbetween the two tissues. One of the mesodermalceilsis pycno~ic(arrow). x 250. FIG. 2. Pycnotic cell in the space between optic vesicle and presumptive lens. The nuclear membrane has partially broken down. x 24500. the thick processes generally have a diameter similar to that of the cell but taper as they extend into the interspace (Fig. 4). Thin processes sometimes extend from the thick processes [Figs 4 (b) and 5 (b), (c)] or m a y extend directly from the bases of the cells [Fig. 5 (a)]. In some cases these processes bridge the interspace and make contact with the opposing tissue [Figs 4(b) and 5 (a), (b), (c)]. In the transmission electron microscope the thick processes are seen to contain large numbers of vesicles, some of which have an electron dense coat on their membrane (Fig. 6). These vesicles probably arise by micropinocytosis because similarly coated pits are also commonly seen in the plasma membrane of the processes. Thin processes that extend from thick processes are largely comprised of fine filaments and contain few, if any, cytoplasmic organelles [Fig. 6(b); see also McAvoy (1980b)]. In addition to the cytoplasmic processes there is a network of fibrils "in the interspace. These are associated with the cytoplasmic processes and the basal surfaces of the presumptive lens and optic vesicle (Figs 4 and 5). Some of these fibrils have a periodicity characteristic of collagen fibrils (McAvoy, 1980). By 12 days of embryonic development the lens placode and optic vesicle have invaginated to form the lens pit and optic cup, respectively (Fig. 7). The interspace averages 5"7+1"6#m so there is essentially no change in width from 11 days. Cytoplasmic processes are not as common as at 11 days but they can still be detected [Fig. 8 (b)]. Mesodermal cells are common in the interspace particularly in the vicinity 16
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FIG. 3. Late in the l l t h day the lens placode (LP) and optic vesicle (OV) begin invagination. Cytoplasmic processes extend across the space between tissues (arrow). A mesodermal cell remains sandwiched between the tissues (open arrow), x 800. Fro. 4 (a), (b). Thick cytoplasmic processes extend from the lens plaeode (LP) and optic vesicle (OV). Fibrils are often associated with the thick processes. In (b) a thin process branches from a thick process and appears to make contact with the optic vesicle. (a) x 4500; (b) x 5000.
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of the foetal fissure. Cells from outside the developing eye have access to the interspace via the foetal fissure and presumably most of the mesodermal cells of the vitreous enter through this passage. Mesodermal cells t h a t are trapped in the interspace between the optic vesicle and presumptive lens m a y also contribute to the cell population of the vitreous since they are present at all stages of early development. However these are few in number and it is not clear if any remain viable.
Fic. 5. (a) Thin processesextend across the space between the presumptive lens (PL) and optic vesicle (OV). The processes in (b) and (c) appear to extend from thick processes (arrows) Fibrils are associated with the processes as well as the basal regions of the epithelial cells. • 6000. The main feature'of the interspace at 12 days is the dense meshwork of fibrils (Fig. 8). These are associated with the mesodermal cells as well as the two cellular layers. In the transmission electron microscope clumps of amorphous material are also detected at this stage. This material is present in the interspace but m a y also be associated with the basal lamina of the presumptive lens and optic cup [Fig. 9 (a), (b)]. The basal laminae of these two tissues is highly elaborated in some regions. This is particularly obvious in the case of the optic cup where it often forms several irregularly arranged layers and extends into the interspace. Thus at least some of the earliest vitreous constituents are laid down by the epithelial ceils lining the interspace. This has also been established in chicks b y Silver and Wakely (1974). By 13 days the lens vesicle has formed and cells in the posterior of the vesicle have commenced to elongate to form the primary lens fibres. The interspace is substantially wider than at 12 days and blood vessels are present often containing blood cells at various stages of maturation (Fig. 10). The endothelial cells of the vessels are said to arise from the mesodermal cells t h a t migrate into the interspace via the foetal fissure (Mann, 1950). The basal laminae of the lens and optic cup are obviously elaborated at this stage. In the lens there are 5-6 layers of basal laminae and together these form the incipient capsule [Fig. 11 (a)]. In the optic cup the basal laminae are not present in regularly arranged layers but, as at 12 days, extend into the interspace [Fig. 11 (b)]. 16-2
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Fio. 6. (a) A thick cytoplasmic process extends across the interspace (IS) from the presumptive lens (PL) to the optic vesicle (OV). (b) The thick cytoplasmic process has a thin process branching from it. Another process closely associated with the thick process (arrow) contains numerous fine filaments. This process may have been attached to the basal lamina of the optic vesicle but appears to have broken aw%v leaving part of it still attached (open arrow). The thick process contains many vesicles some of which are coated vesicles. A coated pit is indicated by the horizontal arrow. (a) x 3500; (b) x 11400. T h e b a s a l c y t o p l a s m of t h e lens cells c o n t a i n s n u m e r o u s c o a t e d pits a n d vesicles [Fig. 11 (a)] s i m i l a r to t h o s e p r o m i n e n t l y f o u n d at 11 d a y s in t h e c y t o p l a s m i c processes. T h e s e are also p r e s e n t in t h e o p t i c cup cells b u t are n o t so a b u n d a n t . E l e c t r o n dense bodies t h a t are a s s o c i a t e d w i t h t h e c o a t e d vesicles are p r o b a b l y lysosomes.
4. D i s c u s s i o n T h e s p a t i a l r e l a t i o n s h i p b e t w e e n o p t i c vesicle, p r e s u m p t i v e lens e c t o d e r m a n d m e s o d e r m was i n v e s t i g a t e d d u r i n g t h e e a r l y s t a g e s of e y e m o r p h o g e n e s i s . M e s o d e r m a l cells are p u s h e d aside as t h e o p t i c vesicle a p p r o a c h e s t h e e c t o d e r m b u t s o m e r e m a i n s a n d w i c h e d b e t w e e n t h e t w o tissues. S o m e of t h e s e o b v i o u s l y d e g e n e r a t e b u t o t h e r s
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FIG. 7. Lens pit (LP) and optic cup (OC) at 12 days. Mesodermal cells are present in the space between these tissues particularly in the vicinity of the foetal fissure (F). • 360. FIG. 8. (a) The interspace at 12 days is filled by a network of fibrils. These connect up with both the mesodermal cells and the epithelial cells. • 2100. (b) Thick (T) and thin (t) cytoplasmic processes are sometimes present. • 2700.
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FIG. 9. The interspace at 12 days contains mesodermal cells and clumps of amorphous material. (a) The basal lamina, particularly on the optic vesicle (OV) side sometimes extends into the interspace (arrow) and (b) forms several layers drawn out into many folds. Amorphous material is often associated with the basal lamina (arrow) (a) x 2800; (b) x l1550. persist until the lens p i t stage. A t this stage m e s o d e r m a l cells from outside the optic p r i m o r d i u m have access to a n d are t h o u g h t to enter, the interspace v i a the foetal fissure (Mann, 1950). I t is n o t possible in this morphological s t u d y to d e t e r m i n e whether all of the sandwiched m e s o d e r m a l cells degenerate, or if a n y of t h e m r e m a i n viable a n d c o n t r i b u t e to t h e composition of the vitreous. However, it is c o m m o n l y assumed in h u m a n s t h a t the vascular elements of the vitreous are formed t o t a l l y from m i g r a t o r y m e s o d e r m a l cells (see Mann, 1950). I t has been suggested from studies on an a n o p h t h a l m i e strain of mice t h a t m e s o d e r m a l cells have an i n h i b i t o r y effect on lens differentiation (Silver and Hughes, 1974). I n these mice viable m e s o d e r m a l cells r e m a i n between the p r e s u m p t i v e lens and optic vesicle a n d are sometimes seen in mitosis, whereas in n o r m a l mice m e s o d e r m becomes necrotic and is resorbed. I n the r a t it is not clear if all the m e s o d e r m a l cells become necrotic during the period of close association of the tissues, i.e. 12th a n d 13th
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FIG. 10. The interspace at 13 days contains many mesodermal cells. Blood vessels have formed and contain blood cells at various stages of maturation, x 2300. Fro. 11. (a) The lens capsule consists of approximately 6-7 layers of basal laminae. Coated pits (arrow) and vesicles are common along with lysosomc:like structures (L). x 28000. (b) The basal laminae of the optic cup (OC) forms several irregular layers. • 10000.
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days of development. Nevertheless viable mesodermal cells, if any, are few in number and obviously do not interfere with the interaction between presumptive lens and optic vesicle/cup. This study shows that during the period of close association between the optic vesicle/cup and presumptive lens there are cytoplasmic processes extending from both tissues. The processes are detected early on the 12th day before the lens placode forms. Processes are also abundant between lens placode and optic vesicle. They are not as common, although still detected between lens pit and optic cup. The processes are not detected after 13 days when the space between the two tissues has widened to form the primary vitreous. The role of the processes is not clear although it is tempting to speculate that they may be important for the transmission of inducing signals between the tissues. Saxen (1977) has proposed that transmission of information falls into two categories; long range and short range. The former is mediated by substances that can diffuse over large distances, whereas the latter depends on cellular contact with the inducer whether it is with an inducing matrix or cells of another type. In the case of the lens some of the cytoplasmic processes make contact with the opposing tissue (see also McAvoy, 1980a). Therefore, on this basis this interaction would be classified as an example of cell mediated induction. However, other experiments and observations do not, in general, support this view. Karkinen-Ji~skel~inen (1978) showed by in vitro transfilter experiments that lens induction took place in chicks even though no cellular contacts were detected by electron microscopy in the filter interposed between ectoderm and optic vesicle. Cellular contacts have not been detected in chicks in vivo despite comprehensive light and electron microscope studies (Hunt, 1961; Weiss and Jackson, 1961; Silver and Wakely, 1974; Hendrix and Zwaan, 1975.) Thus experimental evidence indicates that lens induction can take place in chicks without cell contacts between presumptive lens and optic vesicle. Consistent with this are the m a n y observations that there are no cell processes between these tissues in vivo. On the other hand cytoplasmic processes have been detected in humans (Mann, 1950) and now in rats. Thus the situation with regard to these processes in vertebrates is not clear. They may be present in some species but not all. One difference between chicks and rats is the width of the interspace. In chicks the interspace ranges from 1-3 #m (Hunt, 1961) whereas in rats it averages 5'7 +0"9 #m. Thus in rats and perhaps in other mammals it could be argued t h a t the processes effectively narrow the space between the tissues. In this case cell contact may not be an important event but the processes may be necessary for adequate communication between presumptive lens and optic vesicle/cup during the early period of their interaction. Complete lens differentiation depends on a continuous interaction with the optic cup (see, for example, Le Cron, 1907 ; Muthukkaruppan, 1965) as does the differentiation of epithelial cells into fibres during lens growth (Coulombre and Coulombre, 1963; Muthukkaruppan, 1965; Yamamoto, 1976). This inducer must be a diffusable substance since it can be extracted from the vitreous (Beebe, Feagans and Jebens, 1980) and is present in retina conditioned cell free medium (McAvoy, unpublished results). Consistent with this is the observation that no cytoplasmic processes are present at 14 days when the primary fibres are forming. However this does not rule out the need for cell contact at 11 and 12 days since the inductive signal for fibre formation need not be the same as the signal for the initiation of lens morphogensis (See McAvoy, 1980).
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Another role of the processes m a y be purely mechanical. The cytoplasmic processes together with the fibrils that extend from them and from the basal laminae of the presumptive lens and optic vesicle/cup m a y be important for the adherence of these two tissues. Zwaan and Hendrix (1973) suggested that this adhesion is a prerequisite for invagination. In the chick they showed t h a t the area of contact between presumptive lens and optic vesicle becomes fixed soon after these tissues become associated and suggested that the increased population pressure caused by cell proliferation results in lens cell elongation and finally invagination. Recently Schook (1980) has stressed the importance for invagination of an active extrusion of apical cell cytoplasm in cells of the lens placode. He suggests that because of this extrusion the placode cells which are held together at the apical side, gradually shrink at their apex and grow out in a basal direction. In any case adhesion between presumptive lens and optic vesicle is obviously important in the co-ordination of invagination of lens placode and optic vesicle to form lens pit and optic cup, respectively. A potentially interesting observation is that the thick processes contain an abundance of micropinocytotic pits and vesicles, m a n y of them similar to the coated pits and coated vesicles described by Goldstein, Anderson and Brown (1979). The pits and vesicles are also present in the basal cytoplasm of the lens and optic vesicle cells but are much more abundant in the processes. Coated pits and vesicles have been shown to be involved in receptor-mediated endocytosis, i.e. a process whereby selected extracellular proteins or peptides are first bound to specific cell surface receptors which are clustered in specialized regions of the cell surface and form coated pits. These are internalized to form intracellular coated vesicles. I t is now generally recognized t h a t receptor-mediated endocytosis has a fundamental role in growth, nutrition and differentiation of cells. Consequently during embryonic development it m a y be envisaged t h a t coated pits and vesicles play an essential role, at least in some forms of tissue interactions, in uptake of molecules from associated tissues which m a y determine the fate of the receptive cells. The presence of coated pits and vesicles indicate that the presumptive lens cells are taking up specific environmental signals. This coincides with the initiation of lens morphogenesis and it is tempting to speculate that the presumptive lens cells with the aid of cytoplasmic processes take up an inducer substance from the optic vesicle by receptor-mediated endocytosis. Coated pits and vesicles are also abundant at 13 days in the basal cytoplasm of the cells in the posterior part of the lens vesicle which form the primary fibre cells. I t could be again argued t h a t this represents the uptake of specific molecules involved in the initiation of fibre differentiation, although these are not necessarily the same molecules t h a t trigger lens morphogenesis. Thus it could be concluded that lens induction depends on the displacement of mesodermal cells, which provide cues for skin differentiation, by the optic vesicle which provides cues for lens morphogenesis. The lens cells m a y take up the inducing molecules by receptor-mediated endocytosis particularly in cytoplasmic processes which narrow the space between the interacting tissues. ACKNOWLEDGMENTS I am grateful to Melody Abbott for her technical assistance and to the University of Sydney Electron Miscroscope Unit for assisting me in the use of their Scanning Electron Miscroscope facility. Support for this research was provided by grant no. 1 RO1 Ey 03177 awarded by the N.E.I. Department of Health, Education and Welfare, U.S.A.
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Alexander, L.E. (1937). An experimental study of the role of optic cup and overlying ectoderm in lens formation in the chick embryo. J. Exp. Zool. 75, 41-73. Beebe, D. C., Feagans, D. E. and Jebens, H. A. H. (1980). Lentropin: a factor in vitreous humor which promotes lens fiber cell differentiation. Proc. Nat. Acad. Sci. U.S.A. 77, 490-3. Cohen, A. I. (1961). Electron microscopic observations of the developing mouse eye. I. Basement membranes during early development and lens formation. Dev. Biol. 3. 297-316. Coulombre, J. L. and Coulombre, A. J. (1963). Lens development: fiber elongation and lens orientation. Science 142, 1489. Goldstein, J. L., Anderson, R. G. W. and Brown, M. S. (1979). Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature (London) 279, 679-85. Hendrix, R.W. and Zwaan, J. (1975). The matrix of the optic vesicle presumptive lens interface during induction of the lens in the chicken embryo. J. Embryol. Exp. Morph. 33, 1023-49. Hunt, H. H. (1961). A study of the fine structure of the optic vesicle and lens placode of the chick embryo during induction. Dev. Biol. 3, 175-209. Karkinen-J~gskel~inen, M. (1978). Transfilter lens induction in avian embryo. Differentiation 12, 31-7. Karnovsky, M. J. (1965). A formaldehyde-gluteraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137a. Le Cron, W.L. (1907). Experiments on the origin and differentiation of the lens in Amblystoma. Am. J. Anat. 6, 245-57. Lewis, W. H. (1907). Lens formation from strange ectoderm in Rana sylvatica. Am. J. Anat. 7, 145-69. Mann, I. (1950). The Development of the Human Eye. Grune & Stratton, New York. McAvoy, J. W. (1980a): Induction of the eye lens. Differentiation 17, 137-49. McAvoy, J. W. (1980b). Cytoplasmic processes interconnect lens placode and optic vesicle during eye morphogenesis. Exp. Eye Res. 31,527-34. Muthukkaruppan, V. (1965). Inductive tissue interaction in the development of the mouse lens in vitro. J. Exp. Zool. 159, 269-87. Saxen, L. (1977). Morphogenetic tissue interactions: an introduction. In Cell Interaction in Differentiation (Ed. M. Karkinen-Jgs L. Saxen and L. Weiss). Pp. 145-51. Academic Press, London. Schook, P. (1980). Morphogenetic movements during the early development of the chick eye. An ultrastructural and spatial reconstructive study. A. Invagination of the lens placode. Acta Morphol. Neerl. Scand. lg, 133--57. Sengel, P. (1958). gecherches exp6rimentales sur la diff~renciation des germes plumaires et du pigment de la peau de l'embryon du poulet en culture in vitro. Ann. Sci. Nat. Zool. 20, 431-514. Silver, J. and Hughes, A. F. W. (1974). The relationship between morphogenetic cell death and the development of congenital anophthalmia. J. Comp. Near. 157, 281-302. Silver, P. H. S. and Wakely, J. (1974). Fine structure, origin and fate of extracellular materials in the interspace between the presumptive lens and presumptive retina of the chick embryo. J. Anat. 118, 19-31. Spurr, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43. Wakely, J. (1977). Scanning electron microscope study of the extracellular matrix between presumptive lens and presumptive retina of the chick embryo. Anat. Embryol. 150, 163-70. Weiss, P. and Jackson, S. F. (1961). Fine-structural changes associated with lens determination in the avian embryo. Dev. Biol. 3, 532-54. Yamamoto, Y. (1976). Growth of lens and ocular environment: role of neural retina in the growth of mouse lens as revealed by an implantation experiment. Development, Growth and Differentiation lg, 273-78. Zwaan, J. and Hendrix, R.W. (1973). Changes in cell and organ shape during early development of the ocular lens. Am. Zool. 13, 1039-49.