Ординатура / Офтальмология / Английские материалы / Development of the Ocular Lens_Lovicu, Lee Robinson_2004

24 Michael L. Robinson and Frank J. Lovicu

Figure 1.13. Comparative timetable of lens morphogenesis in frog, chick, mouse, and human. *In frog, developmental stages following fertilisation are dependent on temperature and species. The times stated are only approximate and are based on the developing Xenopus laevis at 22–24◦C.

overlying ectoderm (presumptive corneal epithelium). As the lens vesicle represents an invagination of the surface ectoderm, the basal portions of the cells face outward while the apical portions face the lumen. As a result, components of the basal lamina, secreted by the lens cells throughout life, are deposited externally, encasing the lens in a membranous envelope which through appositional growth acquires more layers and eventually forms the lens capsule. During lens morphogenesis, the anterior capsule thickens more slowly than the posterior capsule, but subsequently anterior capsule synthesis overtakes the remainder, and the anterior capsule becomes several-fold thicker in the mature lens.

Concomitant with the initiation of lens capsule formation, we see the appearance of the first hyaloid vessels of the tunica vasculosa lentis, a vascular net that encompasses the lens and is connected proximally to the hyaloid artery of the retina (Fig. 1.14). The main trunk

Figure 1.14. (A) Blood vessels on the lens prior to atrophy of the vasa hyaloidea propria.

(B) Retrogression of the blood vessels on the lens. (Adapted from Mann, 1928.)

of the hyaloid artery leads into the vasa hyaloidea propria, which branches on to the back of the lens, anastomosing to form the posterior vascular capsule. These vessels lead on to the straight capsulopupillary vessels of the lateral part of the vascular capsule, which once again anastomose with loops of the anterior vascular capsule (pupillary membrane) as well as the choroidal vessels (see Fig. 1.14). The tunica vasculosa lentis is thought to provide nutritional exchange for the lens until the anterior chamber and aqueous humour are operational. With embryonic development, the tunica vasculosa lentis eventually retrogress and disappears. It is usually absent at birth in humans, but in lower vertebrates, such as the rat and the mouse, it persists for a short period postnatally.

The positioning of the cells of the lens vesicle ultimately determines their fate. By the time that the lens vesicle has separated from the overlying surface ectoderm, the posterior cells of the lens vesicle are slightly elongated. The more posteriorly situated vesicle cells facing the optic cup elongate first, advancing ahead of the equatorially placed cells and encroaching on the circular cavity of the lumen, rendering it crescentic. These elongating cells, which constitute the primary fibers, eventually make contact with the apical surface of the anterior lens epithelium, obliterating the lumen of the lens vesicle. The monolayer of anterior cells that face the developing cornea increase in number with the growth of the lens and differentiate into the lens epithelium, assuming a low columnar or cuboidal shape.

26 Michael L. Robinson and Frank J. Lovicu

At this stage of lens development, two populations of highly specialised cells that will maintain a defined spatial relationship with each other throughout life are established. The posterior elongated fiber cells, which constitute the mass of the lens, progressively lose their intracellular organelles and nuclei. In contrast, the anterior cuboidal epithelial cells retain a full complement of organelles. Changes in the pattern of cell proliferation during early lens development play an important role in establishing and maintaining this welldefined lens architecture. In rats and mice, for example, the lens pit and early lens vesicle have an evenly distributed mitotic activity, but as the posterior lens vesicle cells begin to elongate, these cells exit from the cell cycle (McAvoy, 1978; Lovicu and McAvoy, 1999). With continued growth of the lens, only the anterior epithelial cells retain the ability to proliferate, becoming largely restricted to a preequatorial population of epithelial cells, which make up the germinative zone of the lens. With increasing age, a marked decrease in proliferation is also observed in this region. Cells produced in the germinative zone are displaced posteriorly toward the lens equator, giving rise to ordered parallel rows of cells lying in the meridia of the lens, known as meridional rows. As these cells move below the lens equator, into the transitional zone, they elongate into secondary fibers. With the continual addition and elongation of the secondary fibers, the primary fiber mass becomes separated from the epithelial cells anteriorly and from the lens capsule posteriorly, and it now occupies the core of the lens as the embryonic nucleus. As new secondary fibers form, they are added superficially to the previous layer of fibers, establishing the lens cortex. This process proceeds very rapidly in foetal life and at a much decreased rate postnatally. With each successive generation of fibers that are laid down, the cell nuclei migrate forward, establishing a defined ‘bow region’ (most apparent in histological sections) before they are eventually lost through terminal fiber differentiation. As all the lens fiber cells are retained, the entire life history of the lens is conserved in the tissue.

lens determination. We discuss the elements of the current model, the general applicability of the model, and directions for future studies.

2.2.Historical Overview

2.2.1.Methodological Considerations

In order to test Spemann’s model of lens induction and demonstrate the role of the optic vesicle in lens determination, investigators have sought to test both the necessity and the sufficiency of the optic vesicle as a lens inductor. Their experiments have generally taken two forms:

1.separation of the PLE from the influence of the OV either by explantation in vitro, transplantation to a remote site, or ablation of the optic rudiment to determine whether lens differentiation could occur in its absence;

2.transplantation of the OV to remote sites, transplantation of foreign ectoderm to the PLE site, or recombination of tissues with the optic rudiment in vitro to test its ability to induce lens formation in tissues not normally fated to undergo lens formation.

These latter experiments address the sufficiency as well as the specificity of the OV’s inductive properties.

As other investigators began to follow up and expand on Spemann’s intriguing observations, conflicting results soon appeared, and conclusive affirmation of Spemann’s model of lens induction was not immediately forthcoming, although the majority of observations were generally supportive. Reviewing the literature from our current perspective, a number of technical limitations and methodologic issues, common especially among the earlier studies, can be identified as contributing to the often conflicting results.

The bulk of lens induction studies have been done with amphibians, but a number of investigators have used chick and, more recently, mouse embryos to test the generality of the emerging principles. Each organism has its own strengths and limitations as an experimental model. Saha et al. (1989), in their critical review of amphibian lens induction studies, raised the two most serious problems, namely, end point definition and host-donor marking.

Of paramount importance to studies of lens determination are the criteria used to ascertain a positive lens response. The early investigators, whether studying amphibian or chick embryos, relied exclusively on the morphology of the responding tissue. If lens differentiation had proceeded to the point where characteristic elongated lens fibers were present,

Figure 2.1. (facing page) Lens development. The steps of lens development are illustrated diagrammatically using the chick as a representative vertebrate embryo. At the primitive streak stage (A), the PR and PLE are located anterior to the PS. At this stage, early positive inductive signals (arrowheads) travel within the plane of the ectoderm from the neural plate to the outerlying nonneural ectoderm. The transverse section in (B) shows early inductive signals from neural tissue as well as underlying mesendoderm that continue through neurulation (C). After neural tube closure, the optic vesicles grow out from the diencephalon and establish close contact with the PLE (D). At about this same time, NC cells migrating throughout the head produce signals that inhibit lens-forming bias ( ) in head ectoderm outside of the PLE (D and E). Inductive signals from the OV elicit LPL thickening and the subsequent steps in lens morphogenesis and differentiation (E, F, and G). FE, foregut endoderm; LE, lens; LP, lens pit; LPL, lens placode; NC, neural crest; OV, optic vesicle; OC, optic cup; PLE, presumptive lens ectoderm; PR, presumptive retina; PS, primitive streak; RE, retina.

30 Marilyn Fisher and Robert M. Grainger

interpretation was straightforward. Often, however, investigators were confronted with less clear-cut ectodermal thickenings or small vesicles, end points that, while superficially resembling early placode or vesicle stages of lens differentiation, could equally well represent artifacts resulting from the wounding of the epithelium during the experiment. In the case of chick experiments, some authors acknowledged this problem, while others interpreted even ectodermal thickenings as a positive lens response.

McKeehan (1951) was the first to try to circumvent this problem by applying rigorous cytological criteria to define the end points of his experiments. He described a number of cytological changes that normally occur in the PLE of chick embryos from the time the OV first contacts it until there is a well-defined lens placode. These changes include decreased vacuolization of the cytoplasm, reorientation, reshaping, and alignment of the nuclei, features that distinguished the PLE from surrounding ectoderm. A significant advance in this area came when Mizuno and Katoh (1972a, 1972b) introduced the use of antisera to lens crystallins to assay for lens response in their studies of chick lens induction. Since they found crystallins were synthesized already at the placode stage, the presence of these lensspecific proteins served as a reliable indicator that the initial stages of lens differentiation had begun. This same criterion was adopted in amphibian studies beginning with Henry and Grainger (1990). Recently, Sullivan et al. (1998) cautioned against relying exclusively on the immunohistochemical detection of δ-crystallin as the sole indicator of chick lens induction, as δ-crystallin is expressed in some nonlens tissues. They recommended instead an assay combining crystallin expression and cell elongation.

The second technical limitation common to both amphibian and chick lens induction studies was a lack of proper host and donor labeling to enable unambiguous recognition of the source of induced lenses in transplantation experiments. In all vertebrates studied to date, there is extremely tight adherence between the OV and overlying PLE during the stage when most investigators believed the critical inductive interaction was occurring. This tight adherence made clean separation of the OV from the PLE, either for ablation or transplantation purposes, quite difficult, especially when investigators were using purely mechanical separation techniques. Thus, in many cases there was the possibility that a lens found associated with a transplanted OV might actually have arisen from PLE cells carried with the OV rather than from induction of the overlying nonlens ectoderm. This very real possibility was documented by Grainger et al. (1988) in experiments with the frog Xenopus laevis. The problem was not addressed practically in chick studies until KarkinenJa¨askel¨ ainen¨ (1978a) employed the distinctive quail nucleolar organization popularized by Le Douarin (1969) to label grafted tissues in her experiments. In amphibians, apart from a small number of studies that relied on pigmentation differences in interspecies grafts or Nile blue sulfate labeling, the host and donor–marking problem remained until Henry and Grainger (1987) applied the horseradish peroxidase lineage–labeling technique introduced by Jacobson and Hirose (1978) and Weisblat et al. (1978).

A general concern that particularly applies to chick experiments is the great diversity among the experimental approaches of investigators who were trying to answer basically the same questions. Chick embryos present some unique experimental challenges that made certain types of manipulation more difficult than in amphibian embryos. For example, Danchakoff (1926) found that it was very difficult to ablate an OV in a chick embryo employing the same method that Lewis (1904) had used in frog embryos, that is, by lifting a flap of ectoderm, cutting out the underlying OV, and replacing the ectoderm without damaging the PLE. She therefore used a ventral approach to ablate OVs, but she also recognized that in the process she was damaging other tissues that might be (and indeed

have subsequently been shown to be) important for lens induction. Other investigators have neither acknowledged this problem nor have given clear descriptions of how they performed OV ablations.

Similarly, in making transplants of OVs to regions away from the normal lens-forming ectoderm, investigators used everything from half the forebrain of neural plate–stage embryos to fragments of the well-developed optic cup. To circumvent the problem of the strong adherence between OV and PLE, Alexander (1937) cut off the distal part of the OV with the PLE attached and used the remainder, primarily the presumptive pigmented retina and the optic stalk with attached forebrain fragment. The underlying assumption that these diverse starting materials would have the same inductive potential as an intact OV is likely not valid.

Finally, there is much variation in the culture media used for in vitro studies of chick lens induction. Some investigators used solid culture media, while others used a variety of different formulations supplemented with varying amounts of horse, fetal calf, or chick serum in addition to chick embryo extract. All of these variations in methodology can make it difficult to compare or relate the results of different investigators.

2.2.2. Necessity of the Optic Vesicle for Lens Induction

Although Spemann’s (1901) first experiments suggested that the OV was required for lens formation in frog embryos, there soon followed reports of lens formation in the absence of the OV in several amphibian species (King, 1905; Spemann, 1907, 1912) as well as in fishes (Mencl, 1903; Lewis, 1909; see Jacobson and Sater, 1988 for an extensive list). These “free” lenses presumably arose without any contact with the late neural stage OV. While not generally subject to rigorous authentication either as legitimate lenses or as having arisen in the complete absence of eye tissue, they nevertheless cannot all be discounted (discussed by Saha et al., 1989). Spemann concluded from the existence of these free lenses, which in no case resembled normal, fully differentiated lenses, that each species had some degree of innate tendency toward lens formation but that only under the influence of the OV could this lens-forming potential be fully realized (Spemann, 1938).

Experiments involving OV ablations in chick embryos similarly gave mixed results. Danchakoff (1926) and van Deth (1940) reported complete absence of lens formation following OV ablations in embryos from 7–10 and 11–20 somite stages, respectively. In contrast, Waddington and Cohen (1936) reported that slight ectodermal thickenings were present 27 hours after ablation of the OV in 4–5 somite stage embryos. These thickenings may be similar to some of the weaker cases of free lenses in amphibians, and Waddington and Cohen interpreted them as reflecting a “tendency to self differentiation” of lenses. Their interpretation might have been strengthened by waiting longer, as they apparently terminated the experiment at a stage when unoperated controls would be expected to have only a well-defined placode. Somewhat later experiments with chick embryos addressed both the necessity of the OV and the mechanism of its influence on the PLE. McKeehan (1951) blocked contact between the OV and the PLE by interposing a strip of cellophane. He found that lens response was blocked in that part of the PLE that was separated from the OV by cellophane but not in nearby areas where the two tissues were still in contact. In contrast, interposing agar strips did not block induction (McKeehan, 1958; van der Starre, 1977, 1978). In fact, van der Starre (1978) reported that agar strips, after being held in contact with optic cup tissue for 4 hours, could act in place of the OV to induce lens response in head ectoderm of stage-18 embryos (staging according to Hamburger and Hamilton [H&H] 1951).

32 Marilyn Fisher and Robert M. Grainger

Following up on Muthukkaruppan’s (1965) observations on lens development in mice, Karkinen-Ja¨askel¨ ainen¨ (1978a) compared the lens-forming ability of PLE taken from chick and mouse embryos at a stage just prior to contact by the OV. Muthukkaruppan had explanted PLE around the time of initial contact with the OV and found that PLE alone did not form lenses but did so readily when combined with the optic cup, either in direct contact or separated by a millipore filter. In similar experiments, Karkinen-Ja¨askel¨ ainen¨ found a very low percentage of mouse PLE (23%) and no chick PLE formed lenses when cultured alone, but both formed lenses readily (80–90%) when cocultured with the OV, either in direct contact or separated by a millipore filter. Interestingly, Karkinen-Ja¨askel¨ ainen¨ reported that if fetal calf serum was replaced in the culture medium with chick serum, lenses formed in over 50% of isolated, precontact chick PLE samples, and precontact mouse PLE developed recognizable lenses more than 50% of the time if cultured in human amniotic fluid. The combination of morphology and positive crystallin immunohistochemistry suggests that the lens response of isolated PLE in culture is legitimate and argues that, in both chick and mouse, lens determination is well advanced by the time the OV contacts the PLE. Barabanov and Fedtsova (1982) explanted pieces of ectoderm from all over the head to assess lensforming potential and found that between H&H stages 5 and 11 all of the head ectoderm had an equal ability to manifest lens differentiation despite not being in intimate contact with the OV. In contrast, they found that H&H stage 10 trunk ectoderm had no ability to form lenses when isolated in culture. These results have recently been confirmed by C. Sullivan and R. Grainger (unpublished data), who found lens-forming ability in ectoderm explants from H&H stage 8 and 10 embryos even in serum-free medium. Furthermore, Barabanov and Fedtsova (1982) found that non-PLE head ectoderm had lost lens-forming ability by H&H stage 12. The conclusion that can be drawn from the variety of evidence is that the OV is not necessary for the earliest stages of lens development (i.e., placode and vesicle formation) but is necessary for the later stages of development (i.e., the differentiation of the lens vesicle into a mature lens).

2.2.3. Sufficiency of the Optic Vesicle as a Lens Inductor

The other side of the coin with regard to the role of the OV as the primary lens inductor is the question of whether the OV can by itself direct lens development. When Lewis (1904) transplanted OVs from late neurula stage embryos underneath the ectoderm posterior to the PLE and observed lens formation, he concluded that the OV is not only necessary but also sufficient for directing lens differentiation in tissues that would normally never form lenses. There have been many variations on Lewis’s experiment, including experiments in which foreign tissues as diverse as gastrula stage ectoderm and late neurula stage trunk ectoderm were transplanted over various staged optic rudiments as well as experiments in which the OV was transplanted to a variety of foreign locations. The amphibian studies, which were reviewed by Saha et al. (1989), show that there are some circumstances in which the OV apparently induces lens development from non-lens ectoderm, lending support to Lewis’s conclusion. However, when all the results are taken together, it becomes clear that the OV is capable of inducing a lens response in some embryonic ectoderm but not all and that the closer the ectoderm source is to the PLE, the better the lens response.

The results of similar experiments with chick embryos likewise support the conclusion that there are limited circumstances in which the OV is sufficient to induce lenses in nonlens ectoderm. In the chick experiments, there was even more diversity among the sources of ectoderm tested; these sources ranged from primitive streak blastoderm to chorioallantoic

ectoderm of 8- to 9-day-old embryos. Van Deth (1940) reported that the OV from the 20 somite stage embyro had the ability to induce a lens in ectoderm from all regions of a similarly staged embryo when recombined in culture. However, in light of the reliance on a morphologic assay, the absence of host-donor marking, and his own assertion of the difficulty of separating the PLE from the OV during this stage (when they are tightly adherent), one cannot be confident that the observed lentoids and vesicles did not come from PLE cells that contaminated the OV. Alexander (1937) transplanted whole or partial optic cups, free of lens tissue, from 25–40 somite stage embryos to the body wall of hosts ranging from 1 to 13 somites. He concluded that the younger the host and the closer to the normal eye region the transplant is located, the greater the probability of lens formation, although even at best the frequency of lens response remained quite low (15 of 118 cases overall). There are two points to consider here: (1) The young hosts used were at an age when the head ectoderm is already likely to be strongly predisposed toward lens formation (Grainger et al., 1997; Barabanov and Fedtsova, 1982), so it is not surprising that lenses formed close to the eye region (12 of 15 positive cases); (2) most of Alexander’s transplants were partial optic cups rather than whole cups, and most showed some neural and pigmented retinal differentiation by the end of the experiment. Since he used no host-donor marking in these experiments, one cannot rule out the possibility that some or all of the lenses that formed, especially in the trunk region (3 of 15 positive cases), were the result of contamination. Thus, Alexander’s extensive experiments do not provide strong evidence in support of the sufficiency of the optic rudiment as a lens inducer.

McKeehan (1951) also made transplants of the optic rudiment from embryos, primarily at the 6 somite stage, placing them in such locations as the coelom or beneath the ectoderm of the head, neck, or flank. The ages of the hosts are not specified in all cases, but some were as old as 14 somite stage. Using his rigorous cytological criteria for assessing positive lens response, he found only 6 of 29 cases that were positive, and he discounted 4 of these 6 as having arisen from contamination of the graft with donor PLE, since those grafts were taken from donors where the OV and PLE had already formed a strong adherence, making complete separation impossible. In the remaining 2 positive cases, the grafts consisted not of the OV alone but of one-half of the forebrain from embryos at the 4 somite stage. Thus, McKeehan’s results likewise argue against the sufficiency of the OV as a lens inducer.

The strongest case for the sufficiency of the OV as an inducer comes from KarkinenJa¨askel¨ ainen¨ ’s (1978a, 1978b) reports of the ability of the OV to induce lens formation in trunk ectoderm from embryos at 5–14 somite stages (the illustrated case is an embryo at 8 somites). Despite the fact that she used quail and chick chimeric grafts for host-donor marking and crystallin immunohistochemistry as a marker for lens differentiation, she does not use both together in any of the illustrated cases. There is one case where the induced structure looks obviously lenslike and shows positive crystallin immunoreactivity, but it does not have host-donor marking. In another case, the induced structure obviously shows trunk ectoderm origin but does not look lenslike and was not immunostained to demonstrate crystallin expression. Following up on these observations, Karkinen-Ja¨askel¨ ainen¨ (1978b) studied the inductive influence of the OV using trunk ectoderm as a responding tissue. In those studies, she reported that lens response could be elicited in up to 60% of cases, even when the OV and the trunk ectoderm were separated by a millipore filter. However, as in her previous study, here too the illustrated cases do not simultaneously show host-donor marking and crystallin expression, leaving the interpretation open to question. Furthermore,