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Experimental embryology of the vertebrate eye Alfred J. Coulombre The size, shape, position, and orientation of the tissues of the vertebrate eye relative to each other fall within the narrow geometric tolerances compatible with the optical function of this organ. During embryonic development the establishment and maintenance of appropriate relationships among the several ocular tissues remit from an orderly complex of specific interactions among the tissues. Each tissue may be studied as a source of influence on other tissues or, alternatively, as a target of influences arising from other tissues. This paper focuses attention on the lens. The lens may be considered as a target of influences which emanate from the eyecup and neural retina and which are involved in lens induction, lens fiber differentiation, lens suture orientation, lens growth, and the orientation of the lens relative to the optic axis. The lens, in its turn, is a source of influence in the induction of corneal anterior epithelium from ectoderm and in the control of the accumulation of the vitreous substance. The accumulation of vitreous substances importantly influences the size and shape of the pigmented epithelium, choroid coat, and sclera. The analysis of each tissue as both a source and a target of influences permits the construction of flow sheets of tissue interactions in the developing eye. These floio sheets provide a rational basis for understanding the teratology of this organ and represent causal chains which must connect at many points with events at the molecular and chromosomal levels. T to study the developing lens include extirpation, transplantation, explantation, autoradiography, and the confrontation of lens precursors with other tissues. Intervention at specific stages during lens development has revealed many of the successive steps which initiate and channel the differentiation of the lens cells. At the morphogenetic level such studies are beginning to identify and to characterize the mechanisms which continually regulate the position, size, orientation, and shape of the developing lens. During embryogenesis the vertebrate lens is both the target of influences which arise from surrounding tissues and a source of highly specific influences which are essential for the normal differentiation and morphogenesis of some of the other ocular tissues. Enough information has accumulated concerning the influences which operate upon the lens at each stage of development, so that it is possible to con- .he lens of the vertebrate eye has a re±h markably small number of components. Within the acellular fibrous capsule which surrounds it there is a single type of cell and possibly a modest amount of extracellular space. The lens cells are at several stages of cytodifferentiation. In the anterior epithelium they are cuboidal and synthesize proteins of the alpha and beta crystallin groups; at the equator of the lens they begin to elongate and to manufacture proteins of the gamma crystallin group.1'2 In the lens cortex they complete their maturation as lens fibers; then as they become buried deeper in the lens fiber mass they lose their cell nuclei. The experimental techniques which have been used From the Laboratoiy Neuroanatomical Sciences, National Institute of Neurological Diseases and Blindness, National Institutes of Health, Bethesda, Md. 411 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932951/ on 05/10/2017 412 Coulombre Investigative Ophthalmology August 1965 Chorda Mesoderm PRESUMPTIVE NEURAL ECTODERM ECTODERM Notochord• Axial Mesoderm—»-• NEURAL PLATE NEURAL TUBE Mitosis Ventricular »-l PressureW OPTIC VESICLE NON-NEURAL " ECTODERM ^ BASEMENT MEMBRANE Archenteron Roof. »W Heart Mesoderm *-f PRESUMPTIVE LENS ECTODERM PRESUMPTIVE "NEURAL RETINA" LENS PLACODE VITREOUS COMPONENTS LENS CUP Cell Death LENS VESICLE- 1 ' LENS FIBERS LENS EPITHELIUM LENS CAPSULE Fig. 1. This flow sheet represents the major steps in lens development (heavy arrows) and indicates some of the influences (light arrows) which impinge on, or are exerted by, the embryonic lens. Dotted lines indicate uncertain relationships. struct a flow sheet (Fig. 1) which (1) tentatively delineates the sequence of individual steps in the differentiation and morphogenesis of this structure; (2) focuses attention upon specific gaps in our knowledge of the developing lens system; and (3) provides a rational basis for understanding defects of the lens and of the eye which arise from interference with specific steps in the chain. The lens as a target A. Induction. The lens is a target of outside influences even in its origin, which deserves brief recapitulation here. Competence to form lens, which is widespread in the primitive embryonic ectoderm,3 becomes restricted to the head region under the influence of the foregut roof and the presumptive heart mesoderm.3'10 The pre- sumptive neural retina at the tip of the optic vesicle plays the definitive role in the induction of lens from overlying ectoderm.11"17 This induction requires proximity but not contact of the optic vesicle and the overlying ectoderm.is"22 Lens induction continues for a relatively prolonged period during development,23"25 and the ability of the neural retina to support the regeneration and growth of the lens persists even in the adults of some salamanders.20 During its earlier stages the neural retinal influence results in a palisading of the ectodermal cells to form a lens placode,17 which invaginates to form the lens cup. Closure of the edge of the lens cup cuts off the lens vesicle internally, re-establishes continuity in the overlying ectoderm (presumptive corneal epithelium), and completely envelops the lens vesicle in Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932951/ on 05/10/2017 Volume 4 Number 4 Experimental embryology of vertebrate eye 413 the basement membrane (primitive lens capsule) which had underlain the lens placode.27 B. Lens fiber differentiation. The cells of the lens vesicle which lie toward the optic cup elongate progressively to form lens fibers which eventually touch the posterior surface of the lens epithelium and obliterate the cavity of the lens vesicle. This elongation to form lens fibers is an uninterrupted continuation of the cell elongation which began during palisading in the placode. The cells of the lens vesicle which lie away from the optic cup do not elongate but form a simple cuboidal epithelium which covers the fiber mass anteriorly and is continuous with it at the equator of the lens. From the outset mitotic activity ceases in the lens fibers and is confined to the lens epithelium. The cells near the equatorial edge of the lens epithelium develop larger nuclei, more prominent nucleoli, a higher concentration of cytoplasmic RNA, more numerous mitochondria, and increased height. It is in the equatorial region that epithelial cells are progressively recruited into the lens fiber mass. As the lens grows, the axes of the cells of the equator rotate through 90 degrees and take their place at the periphery of the contiguous fiber mass. At later stages the lens fibers which are generated at the equator grow around the previously deposited fiber mass until they meet anteriorly and posteriorly with other fibers in planes of junction which are called sutures.28 C. hens sutures. The patterns of suture planes vary from a single plane to many radiating planes of conjuncture depending on the species. The orientation of these planes with respect to the eye are constant in a given species and become determined during a specific period in embryonic development.20 Prior to this time, experimental rotation of the embryonic lens about its axis results, during ensuing development, in the appearance of suture planes which are normally oriented relative to the eye. Experimental rotation of the lens about its axis following this critical period in embryonic development results in the development of suture planes which are inappropriately oriented with respect to the eye because their orientation has become fixed with respect to the lens. D. Lens growth. At any stage in development the volume of the lens is given by the relationship: V = N7+S+C, where V is lens volume, N is the number of lens cells, v is the mean volume of the lens cell, S is the volume of the extracellular space, and C is the volume of the lens capsule. Both S and C make extremely small contributions to the total lens volume at all stages during development and will not be considered further in this treatment. There is no information currently concerning the volume of individual cells (v) as a function of their position in the epithelium, the equatorial region, or at various depths of the fiber mass, or as a function of developmental time. We do know, as mentioned, that the influence of the neural retina is necessary for the increase in cell volume which occurs when lens cells are transformed into lens fibers. This transformation is of central importance in the growth of the lens, since the lens fiber mass makes the largest single contribution to the increase in lens volume. When the neural retina is removed from the eyes of four-day chick embryos, the growth of the lens ceases in a short time. When the same operation is performed, but a small piece of neural retina is reintroduced into the eye cavity, subsequent growth of the lens is intermediate between that in eyes without neural retina and that in untreated eyes.30 The growth of the lens appears to be independent of the growth of all other tissues of the eye with the exception of the neural retina. By inserting drainage tubes through the eye wall of the four-day chick embryo, a portal of egress was provided for the vitreous substance as it was produced. As a consequence of this operation the eye as a whole grew more slowly than normally Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932951/ on 05/10/2017 414 Coulombre and became microphthalmic. The neural retina continued to grow independently and was thrown into increasingly more complex folds. The outer coats of the eye (cornea, sclera, choroid coat, and pigmented epithelium) grew at rates which were markedly below normal. Despite the subnormal growth of the superficial coats of the eye, the lens grew at a normal rate.31 Not only can the growth rates of the lens and the outer coats of the eye wall be uncoupled in this manner, but the presence of the outer coats does not seem to be essential for normal growth of the lens provided that the neural retina is present in sufficient quantity. For example, removal of the cornea at five days of incubation in the chick embryo has no detectable effect on the growth, shape, or internal organization of the developing lens. At any stage in development N is exhaustively described by the following function: N = n+(M+mr-mo-D)t, where n is the initial number of cells in the lens primordium; M is the number of cells added to the population per unit time as a result of mitosis; m-, is the number of cells added to the population per unit time as a result of migration of cells into the lens population from other tissues; m0 is the number of cells lost to the cell population per unit time as a result of migration of cells out of the lens population; D is the number of cells lost to the cell population per unit time as a result of cell death and dissolution; and t is time. In all but certain experimental situations the terms mx and m0 have no bearing, and can be eliminated from the equation as long as the capsule is intact and the lens remains a closed system. Mitosis is confined to the cells of the lens epithelium and does not occur, at any stage in development, in cells which have traversed the equatorial region to become part of the lens fiber mass. The mitotic activity must be so balanced with respect to the other factors affecting cell number and cell volume that cells are generated at a rate just sufficient to account for the addition of new fibers on the Investigative Ophthalmology August 1965 one hand and the increase in the number of epithelial cells on the other. It is well worth emphasizing that throughout development the lens epithelium has an area which is precisely appropriate to the size of the underlying fiber mass. In the normally developing lens it neither buckles nor becomes so stretched that gaps develop. The contribution of D to the growth of the lens deserves more attention than it has received. It is known that in all but the earlier stages of development the cells at the center of the fiber mass (lens nucleus) lose their cell nuclei and, in this sense, die. However, it remains to be established whether or not any of these cells undergo dissolution followed by removal of the breakdown products from the lens. Despite the fact that mitosis continues in the lens epithelium throughout life in most species, and that fibers continue to be added to the lens cortex at the equator, the increase in the volume of the lens ultimately becomes negligible. This paradox could be resolved by a compensatory loss in cell volume in the mature fibers (the widely held concept of central compression), a compensatory loss of cells, or both. The value of n is a function of the size of the cells in the presumptive lens ectoderm at the time of lens induction, and of the area of contact between the tip of the optic vesicle and the overlying ectoderm.17' 32'3'1 The number of cells in the primitive lens population is not constant; rather, this population is increasing from the very outset. The size of the eye which develops is directly correlated with the area of contact between the vesicle and the ectoderm, and therefore with the number of cells in the lens placode.10'34 However, since the area of the neural retina also depends upon the area of this contact,35' 3G it is impossible to say whether the initial size of the lens population has any importance independently of the role played by the neural retina. During later development there is, in experimental situations, a correlation between the size of the lens Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932951/ on 05/10/2017 Volume 4 Number 4 Experimental embryology of vertebrate eye 415 and the surface area of the neural retina.34 The simple algebraic formulations given previously are accurate, but they can be misleading unless it is realized that each of the terms can, and probably does, vary as a function of developmental time. These relationships focus attention on the major components which contribute to lens size, and invite attention to the mechanisms which control the rate at which each of the components increases during embryonic development. E. Orientation of the lens. Given the optical function of the lens, and the stringent geometric limits set by the laws of optics, this structure can function effectively only if it is appropriately oriented with respect to the optic axis and suitably aligned with the other dioptric media. It is a matter of observation that the lens is usually properly positioned and oriented throughout its entire development. The initial alignment of the lens with the eyecup is assured because its definitive induction is earned out in the zone of contact between the presumptive lens ectoderm and the tip of the optic vesicle. It has been demonstrated in a number of ways that this mechanism continues to operate and continuously adjusts the orientation of the lens as development proceeds. If, in certain salamanders, the iris with its subtended lens is surgically removed and reversed so that its anterior surface now faces the vitreous body, it will heal in place. Subsequent removal of the lens from such animals is followed by the regeneration of a lens from the dorsal iris. The regenerated lens is oriented appropriately with respect to the eye and in reversed orientation with respect to the iris.30 The system which orients the lens during lens regeneration is probably similar to that which has been demonstrated during normal development in the chick embiyo. If the lens of the five-day chick embryo is removed from the eye, rotated 180 degrees, and replaced in such a manner that the lens epithelium now faces the vitreous body rather than the cornea, there is a Fig. 2. This is an axial section of an eleven-day lens of a chick embryo which had been reversed at five days of incubation. The polarity of the lens has become reversed, a new epithelium is being reconstituted anteriorly, and a new fiber mass is developing posteriorly from the original lens epithelium. The lens is of a size, shape, position, and orientation appropriate to its age. complex reorganization of the lens cells. The old lens fiber mass, which is now situated anteriorly, stops growing. The lens epithelium, which is now placed posteriorly, rapidly increases in height as all its cells differentiate into lens fibers. The lens epithelial cells close to the equator continue to divide and reconstruct a new lens epithelium anteriorly at the same time that they contribute fibers to the new fiber mass which has developed from the origi- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932951/ on 05/10/2017 416 Coulombre nal epithelium. As a consequence, the polarity of the lens is reversed 180 degrees (Fig. 2). During this process the lens continues a normal increase in volume, and, despite the complex changes occurring internally, maintains an appropriate shape and orientation.37 If a fiveday lens is removed and replaced with two five-day lenses, the ensuing changes obey the following rules regardless of the orientation in which the lenses are introduced: (1) all of the lens epithelial cells which are on the vitreal side of a plane passing through the ciliary processes elongate to form lens fibers; (2) the lens epithelial cells located on the corneal side of this plane continue to divide, form lens epithelium on the entire corneal surface of the combined lens mass, and reconstitute new equatorial zones adjacent to the ciliary processes, which contribute fibers to the new fiber mass. In the process the combined lens mass achieves an appropriate internal organization (epithelium toward the cornea and lens fiber mass toward the vitreous body), and gradually becomes regulated in its combined shape, orientation, and total volume until these properties match those appropriate to a single lens of the host age. Even if the embryonic lens is replaced not with a whole lens but with an isolated embryonic lens epithelium attached to its adjacent capsule, a reasonably well-shaped lens will develop in which the epithelium invariably faces the cornea and in which the lens fiber mass lies toward the vitreous body. This occurs regardless of the initial orientation of the implant. Even after a six-day chick embryonic lens epithelium has been maintained for five additional days in culture, it will reform a reasonably good and appropriately oriented lens if it is returned to a lentectomized eye, but will not do so if it is transplanted into the coelom of a host embryo.3S This experiment provides a convincing demonstration of the specificity of the eye environment. It has recently been demonstrated in the mouse that the retinal factor(s) In vcstigative Ophthalmology August 1965 which contribute to this mechanism can act across a Millipore filter, and are, therefore, probably diffusible.39 This system appears to have a relatively low taxonomic specificity. If the lens of the five-day chick embryo is replaced with an isolated lens epithelium and capsule from a mouse embryo of 18 days' gestation, the host eye supports the differentiation of the implant into a well-formed lens that becomes appropriately oriented with respect to the chick eye. The lens as a source of influence During an orderly morphogenesis the tissues of the vertebrate eye develop sizes, shapes, positions, orientations, and cellular structures which, in the aggregate, admirably suit this organ for optical function. It is becoming increasingly clear that this highly coordinated development rests upon a complex of prolonged interactions among the tissues of the eye, which assures that they will develop appropriate relationships. It has been fruitful to consider each tissue of the eye not only as a target for influence from surrounding tissues, but also as a source of influences which help to control the development of one or more of the other ocular tissues in highly specific ways. Two influences which the lens exerts deserve mention. A. Induction of the anterior corneal epithelium. The ectoderm which remains on the surface overlying the embryonic eye following closure of the lens pore is destined to form the anterior epithelium of the cornea. At first, competence to form the cornea is widespread throughout the embryonic ectoderm. The lens vesicle, and later the lens, provides the principal inductive influence which assures that the cornea will differentiate over the eye.40"49 The precise alignment of the cornea with the lens is achieved and maintained because of the continuing inductive relationship between the lens and the comeal anterior epithelium.11' "• 40>4S"50 The ability of the lens to induce cornea is gradually lost with age.515'1 Concomitantly the Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932951/ on 05/10/2017 Experimental embryology of vertebrate eye 417 Volume 4 Number 4 anterior epithelium of the cornea becomes increasingly independent of its inductor.2r>> -10, ii, 4a, 4-1, T.), 54-5G j n s o m e forms, however, this dependence persists throughout life. B. The control of the accumulation of vitreous substance. Experiments in the chick embryo suggest that the presence of the lens is necessary for the normal accumulation of the vitreous substance. Removal of the chick embryonic lens at five days of incubation terminates the accumulation of vitreous substance. If the lens is removed from the eye and immediately replaced, the accumulation of vitreous substance is normal. If the lens is removed, boiled, and returned to the eye, vitreous substance does not accumulate. The gross increase in the size of the eye during development is attributable in large part to the accumulation of vitreous substance within the eye.57> 5S For this reason the control of the growth of the vitreous body by the lens has important consequences in that this growth, in its turn, is a necessary condition for the normal growth of the cornea,59'60 the sclera,57 the choroid coat, and the pigmented epithelium.G1 The mechanism by which the accumulation of vitreous substance is controlled by the lens remains to be explored. Conclusion The complex interactions of the ocular tissues during development, in which the neural retina plays a key role, assure an orderly organogenesis. We know an increasing amount concerning which tissues exert influences, during what period they influence, which tissues are competent to respond, and in what ways they respond. Despite this increasing body of information, we do not know the physical basis of any of these numerous influences. The most economical hypothesis is that diffusible substances are released from specific tissues of origin according to a precisely programmed sequence, and that these substances alter the number, size, or state of differentiation of cells in target tissues which are competent to respond. Yet the matter is not so simple. We know, for example, that mechanical tension generated by one tissue can alter the development of a neighboring tissue. We have seen something of the role that the lens plays in this complex of interactions both in the influence which it exerts on surrounding tissues, and in the responses which it gives to specific stimuli from its neighbors. Each tissue of the eye, in its turn, may be analyzed with this same approach. The flow sheet of interactions which emerges from such analyses provides not only a rational basis for understanding the teratology of this organ, but also represents a causal chain which must connect at many points with the events taking place at the chromosomal and molecular levels. REFERENCES 1. Papaconstantinou, J.: The formation of gamma-crystallins during lens fiber differentiation, Am. Zool. 4: 279, 1964. 2. Takata, C , Albright, J. F., and Yamada, T.: Gamma-cristallin and lens fiber differentiation —an immunofluorescent study of amphibian lens regeneration, Science. In press. 3. Mangold, O.: Das Determinationsproblem. III. 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Philpott, G., and Coulombre, A. J.: Lens development. II. The differentiation of embryonic chick lens epithelial cells in vitro and in vivo, Exper. Cell Res. In press. Muthukkaruppan, V.: In vitro studies on embryonic lens induction in tlie mouse, Am. Zool. 4: 278, 1964. Lewis, W. H.: Experimental studies on the development of the eye in amphibia. II. On tlie cornea, J. Exper. Zool. 2: 431, 1905. Durken, B.: Ueber einseitige Augenextirpation bei jungen Froschlarven. Ein Beitrag zur Kenntness der echten Entwicklungs Korrelationen, Ztschr. f. wissensch. Zool. 105: 192, 1913. Fischel, A.: Ueber Rucklaufige Entwicklung. I. Die Ruckbildung der Transplantierten, Arch. Entwcklngsmechn. Organ. 42: 1, 1917. Fischel, A.: Ueber den Einfluss des Auges auf die Entwicklung und Erhaltung der Hornhaut, Klin. Monatsbl. Augenh. 62: 1, 1919. Groll, O.: Uber Transplantation von Ruckenhaut an Stelle der Conjunctiva bei Larven von Rana fusca (Rosel), Arch. Entwcklngsmechn. Organ. 100: 385, 1923. Reyer, R. W.: An experimental study of lens Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932951/ on 05/10/2017 Volume 4 Number 4 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. Experimental embryology of vertebrate eye 419 regeneration in Triturus viridescens. II. Lens development from the dorsal iris in the absence of the embryonic lens, J. Exper. Zool. 113: 317, 1950. Ignatieva, G. M.: Histological change of skin transplanted into the eye in tailless amphibians, Doklady Akad. nauk. SSSR 81: 701, 1951. Ophth. Lit. 5: Abst. 6028. Ignatieva, G. M.: Formation of the cornea in the tadpole from the skin of Triton taeniatus larvae, Doklady Akad. nauk. SSSR 82: 167, 1952. Neifach, A. A.: Investigation of the role of the optic rudiment in the development of the cornea in the chick, Doklady Akad. nauk. SSSR 75: 141, 1950. Neifach, A. A.: Transplantation of the cornea at various stages of development in chorioallantois, Doklady Akad. nauk. SSSR 85: 1177, 1952. Ophth. 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