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

Figure 9.1. Stages in the differentiation of a fiber cell. (A) Mitotically active cell near the equatorial margin of the lens epithelium. (B) Elongating fiber cell. (C) Maturing fiber cell.

(D) Mature fiber cell.

1992; Bassnett and Beebe, 1992; Bassnett, 1995, 1997; Bassnett and Mataic, 1997). In addition, the membranes of adjacent fiber cells partially fuse just before organelles are degraded (Shestopalov and Bassnett, 2000a). Therefore, during the process of terminal differentiation, fiber cells pass through specific programmed stages. Although these stages are not always marked by gross morphological alterations, they can be discerned by the presence or absence of characteristic biochemical markers.

We have divided the life of a fiber cell into four distinct stages (Fig. 9.1). Fiber cell precursors are produced by mitosis of cells in the germinative zone of the epithelium. Fiber cells withdraw from the cell cycle and elongate from columnar progenitor cells at the lens equator. Elongating fiber cells have relatively smooth membranes with distinct adherens junctions near their apical and basal tips. Elongating fibers make direct contact with the capsule and the epithelium at their basal and apical surfaces, respectively. When elongation is complete, fiber cells lose contact with the capsule and the epithelium. Distinct basal and apical adherens complexes can no longer be visualized by antibody staining. These

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maturing fiber cells appear to restructure their lateral membrane complexes, and there is a concomitant increase in the folding of their lateral membranes (Kuszak et al., 1980; Willekens and Vrensen, 1982; Beebe et al., 2001), presumably more firmly locking the fibers to their neighbors. At this point, fiber cell lateral membranes partially fuse, creating large pores between adjacent cells (Shestopalov and Bassnett, 2000a). The final stage in the life of a fiber cell begins with the abrupt loss of intracellular, membrane-bound organelles. The resulting mature fiber cell is the fully differentiated cell type of the lens.

9.3.Organization of Cells at the Lens Equator

9.3.1.The Germinative Zone

In the adult lens, mitotic activity is rare in the epithelial cells. It is only near the lens equator, at the boundary between the epithelium and the fiber cells, that a significant concentration of mitotic activity is seen. This region is termed the “germinative zone.” The cells within the germinative zone are adjacent to the nonpigmented cells of the ciliary processes. It has been speculated that growth factors from the ciliary epithelium are responsible for stimulating the proliferation of germinative zone cells, although this hypothesis has not been tested directly (Schlotzer-Schrehardt and Dorfler, 1993).

Proliferating germinative zone cells can be labeled in vivo by intraperitoneal injection of precursors of DNA synthesis, such as 3H-thymidine or 5-bromo-2 -deoxyuridine (BrdU). Typically, labeled cells from the germinative zone move into the early fiber cell population within a few days to a few weeks. In adult mice, at least 95% of these differentiating cells are daughter cell pairs (Rafferty and Rafferty, 1981). That is, both of the daughter cells of a mitotic division leave the germinative zone and differentiate into fiber cells together. Examination of data from Rafferty and Rafferty (1981) reveals that a similar number of labeled cells remain in the germinative zone after cell division as leave to become fibers. Since nearly all the cells that leave the germinative zone are daughter cell pairs, this observation suggests that those labeled cells remaining in the germinative zone are also daughter cells. If this interpretation is correct, it is reminiscent of the fate of dividing cells in the basal layer of the corneal epithelium. In that tissue, the daughter cells of a mitosis either both move out of the basal layer at the same time and differentiate together or both remain in the basal layer where they may divide again (Beebe and Masters, 1996). The factors that determine whether a pair of daughter cells remains in the germinative zone or leaves to begin the process of fiber cell differentiation are unknown.

From extant data it is not possible to determine whether there is a population of cells that resides in the germinative zone throughout the life of the lens or whether all germinative zone cells eventually differentiate into fibers and are replaced by cells from the adjacent peripheral epithelium. Further studies of the fates of dividing cells in the germinative zone are needed to clarify the pattern of mitosis and migration in this region of the lens and provide insight into the stability of this cell population.

9.3.2. The Transitional Zone

After leaving the germinative zone, cells move into the “transitional zone,” a region in which cells are postmitotic but have not yet begun to form lens fibers. The transitional zone is about 6 or 7 cells wide in adult mice (Rafferty and Rafferty, 1981) and approximately 17 cells wide in adult rats (McAvoy, 1978a). In birds, the comparable region of postmitotic

cells, the “annular pad” (see chap. 1), contains a much larger population of postmitotic cells. These cells form a thickened band of elongated epithelial cells that probably helps to cushion the lens during accommodation, since the ciliary apparatus of birds compresses the lens to increase its focal power.

The forces responsible for the movement of cells from the germinative zone into the transitional zone have not been examined directly in the intact lens. Because all lens cells are part of a continuous epithelial layer, one plausible view is that it is the increase in cell number resulting from mitosis in the germinative zone that causes cells to be displaced posteriorly. Consistent with this view, when cell division in the frog lens is blocked by removal of the pituitary gland, movement of cells through the transitional zone into the fiber mass stops (Hayden and Rothstein, 1979). Furthermore, restoration of lens epithelial cell division by repeated injection of growth hormone (somatotropin) or somatomedin-C (insulinlike growth factor-1 [IGF-1]) restores cell migration and fiber cell differentiation (Rothstein et al., 1980; Klein et al., 1989). However, it is possible that removal of the pituitary gland and/or supplementation with somatotropin or IGF-1 can influence fiber differentiation directly.

Other studies have suggested that factors that stimulate fiber cell differentiation may also cause cells to actively migrate toward the posterior of the lens. Treatment of rat lens epithelial cells with low concentrations of fibroblast growth factors (FGFs) stimulates cell proliferation, while higher concentrations cause cell migration, and still higher concentrations result in fiber cell differentiation (McAvoy and Chamberlain, 1989). This observation led to the hypothesis that mitosis in the germinative zone, migration into the transitional zone, and eventual differentiation into fibers is a response to a standing gradient of FGFs in the ocular fluids. Measurement of FGF levels in the aqueous humor that bathes the anterior epithelium and in the vitreous body adjacent to the lens fibers supports this view (Schulz et al., 1993). The presumed intermediate concentration of FGFs in the ocular fluids adjacent to the germinative and transitional zones could, therefore, stimulate migration from the germinative zone into the transitional zone.

Data from the frog suggesting that mitosis drives cell migration and data from the rat suggesting that cell migration may be regulated directly by growth factors in the ocular media have not been reconciled satisfactorily. It has not been possible to block cell proliferation in the rat lens in vivo (Klein et al., 1989), and to our knowledge the concentration of fiber differentiation factors has not been measured in the intraocular fluids of the frog eye after hypophysectomy.

The mechanism responsible for the cessation of cell division that occurs as cells move into the transitional zone is not well understood. As described above, in vitro experiments suggest that high concentrations of FGFs cause lens epithelial cells to stop dividing and to differentiate (McAvoy and Chamberlain, 1989). Lens epithelial cells also withdraw from the cell cycle and differentiate into fiber cells in transgenic animals that overexpress FGFs in the lens (Lovicu and Overbeek, 1998; Robinson et al., 1998). It is possible, therefore, that activating a “fiber cell differentiation program” is sufficient to assure that cells will exit from the cell cycle. Alternatively, FGFs or other fiber differentiation factors may block lens cell proliferation and trigger fiber cell differentiation independently.

Studies in chicken embryos have demonstrated an activity in the vitreous body that prevents lens cells from dividing in response to growth stimulatory factors present in the anterior chamber (Hyatt and Beebe, 1993). Similarly, when chicken embryo lenses are rotated so that the rapidly dividing epithelial cells are exposed to the vitreous humor, these cells withdraw from the cell cycle within nine hours (Zwaan and Kenyon, 1984). It is not

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evident from these studies whether the antimitotic activity present in the vitreous humor is caused by a fiber differentiation factor or another factor that assures that cells in the posterior of the lens do not proliferate.

Intracellular proteins that play critical roles in the regulation of cell proliferation elsewhere in the body have likewise been implicated in the cessation of cell proliferation in the lens. Studies in mice showed that the cyclin-dependent kinase inhibitor proteins p27KIP1 and p57KIP2 and the retinoblastoma protein (pRb) are needed for lens fiber cells to withdraw from the cell cycle (see chap. 8). Progression through the cell cycle requires the phosphorylation of cyclins by cyclin-dependent kinases. The cyclin-dependent kinase inhibitor protein p57KIP2 inhibits this process, thereby blocking cell cycle progression. p57KIP2 is expressed in cells of the transitional zone and the youngest lens fibers, where it is found bound to cyclins (Gao et al., 1999; Lovicu and McAvoy, 1999). In lenses that lack the gene for pRb, a well-known regulator of the cell cycle, p57KIP2 mRNA levels are low and lens fiber cells do not withdraw from the cell cycle (Morgenbesser et al., 1994; Fromm and Overbeek, 1996). This finding suggests that pRb is required for the expression of p57KIP2 and that at least one function of p57KIP2 is to maintain lens fiber cells in the nonproliferating state by complexing with cyclins. Targeted deletion of both of the cyclin-dependent kinase inhibitors expressed in the lens, p27KIP1 and p57KIP2, leads to continued lens fiber cell proliferation, even in the presence of pRb, confirming that these molecules play an important role in the withdrawal of fiber cells from the cell cycle (Zhang et al., 1998). Lenses lacking both p27KIP1 and p57KIP2 do not synthesize detectable levels of crystallins, suggesting that the function of these molecules is also required, directly or indirectly, for normal gene expression during fiber cell differentiation (Zhang et al., 1998). Other than a requirement for pRb, the mechanisms that link fiber differentiation factors outside the lens cells to the increased expression of p27KIP1 and p57KIP2 within lens fiber cells have not been determined.

9.3.3. Studying Fiber Cell Differentiation in Vitro

The events that occur during fiber differentiation can be described in the intact lens, but studying the underlying mechanism(s) is more easily accomplished in cultured lens epithelial cells stimulated to differentiate into fiberlike cells in vitro. Epithelial explants (whole or partial sheets of lens epithelium) obtained from neonatal rats and chicken embryos have provided the most useful models for these studies.

Dissociated lens cells have also been used to study aspects of fiber cell differentiation in vitro (Menko et al., 1984; Blakely et al., 2000). However, these systems seem less relevant to the in vivo situation because the cells are no longer attached to their basal lamina, the lens capsule. Dissociated lens epithelial cells are also isolated from their neighbors and are usually cultured in serum-containing medium. Dissociation often alters the behavior of epithelial cells, and culture in serum-containing medium can be sufficient to trigger fiber cell differentiation, making it difficult to use such a system to study those factors that normally trigger this process in vivo.

Extended culture of dissociated lens epithelial cells may lead to the formation of “lentoid bodies,” which are aggregates of fiberlike cells. The cells in lentoid bodies can be compared with nearby flattened epithelial cells as a means of studying the properties of “fiberlike” and “epithelial-like” cells in the same culture environment (Berthoud et al., 1999; Blakely et al., 2000; Ibaraki et al., 1996; Menko et al., 1984; Wride and Sanders, 1998).

In explanted chicken embryo lens epithelia, cells elongate and show increased rates of transcription and protein synthesis when cultured in media containing vitreous humor

(Beebe et al., 1980), fetal calf serum (Philpott and Coulombre, 1965; Piatigorsky et al., 1972; Piatigorsky et al., 1973a; Milstone and Piatigorsky, 1975; Milstone et al., 1976), or other differentiation factors (Beebe et al., 1987). These changes are evident within the first five hours in culture. Since differentiating fiber cells are exposed to vitreous humor in vivo, these responses are likely to replicate those occurring in the intact lens.

Cell elongation, increased RNA and protein synthesis, and specialization for crystallin synthesis have not been examined in the same detail in neonatal rat lens epithelial explants. However, from those studies that have been reported, it appears that these processes do not occur as rapidly in rat lens epithelial cells as they do in chicken lens epithelial explants treated in a similar fashion (Campbell and McAvoy, 1984; Peek et al., 1992). For example, in the rat system, cell migration begins at 1 hour; nucleolar swelling, a sign of increased protein synthesis, is seen at 16 hours; and increased α-crystallin mRNA accumulation is detected by 24 hours (Walton and McAvoy, 1984; Peek et al., 1992). It is possible that the slower rate of response seen is due to the fact that cultured rat lens epithelial cells have been studied only postnatally, while in chickens only embryonic lens epithelia have been examined. Fiber cell differentiation may occur more rapidly in tissues from embryos. Consistent with this possibility, a decrease in the rate and extent of fiber cell differentiation has been described in rat lens epithelial explants with increasing postnatal age (Richardson and McAvoy, 1990; Richardson et al., 1992).

There is another fundamental difference in the response of lens epithelial explants from chicken embryos and adult rat lenses to agents that stimulate fiber cell differentiation. Cell elongation in chicken embryo explants occurs perpendicular to the lens capsule, and the elongating cells maintain their attachments to the capsule and to their neighbors (Philpott and Coulombre, 1965; Piatigorsky et al., 1972). In rat lens epithelial explants, cells initially detach from both the capsule and their neighbors and migrate on the surface of the epithelial monolayer (McAvoy, 1988). They subsequently pile up and elongate perpendicular to the capsule (Walton and McAvoy, 1984). In spite of these differences, the fiberlike cells that eventually differentiate in these in vitro systems resemble authentic lens fibers in many ways. For example, the differentiating cells eventually degrade their organelles in a similar fashion to that observed in vivo (Piatigorsky et al., 1973a; McAvoy and Richardson, 1986).

9.4. The Initial Events in Lens Fiber Cell Differentiation

Because chicken embryo lens epithelial cells respond quickly to stimuli that cause fiber differentiation, this section concentrates on studies using this model. Where appropriate, examples from rodent lens epithelia are included.

Treatment of embryonic day 6 (E6) lens epithelial cells with vitreous humor, fetal calf serum, or insulin leads to a rapid increase in the methylation of the membrane phospholipid phosphatidylethanolamine (Zelenka et al., 1982). The addition of three methyl groups converts phosphatidylethanolamine to phosphatidylcholine. Remarkably, the synthesis of phosphatidylcholine by methylation reaches a peak within 6 seconds after addition of chicken embryo vitreous humor or fetal bovine serum. The newly synthesized phosphatidylcholine is degraded by 15 seconds after addition. Insulin, which also stimulates chicken embryo lens cells to elongate, exhibits a slower time course, with phosphatidylcholine being synthesized and degraded within one minute. The slower time course in response to insulin may be caused by the binding of insulin to the endogenous insulinlike growth factor receptor rather than the insulin receptor, since both receptors are present in the lens and insulin binds to the

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insulinlike growth factor receptor with lower affinity than does IGF-1 (Beebe et al., 1987; Alemany et al., 1990; Bassnett and Beebe, 1990). Inhibitors of phospholipid methylation block cell elongation with a dose-response relationship that parallels their ability to reduce phospholipid methylation (Zelenka et al., 1982). In these studies, it was not possible to determine whether the rate of phospholipid methylation and degradation remains elevated after stimulation or whether it rapidly returns to the same level as before stimulation.

Little more is known about the events that follow the synthesis and degradation of phosphatidylcholine in response to vitreous humor or other factors that stimulate fiber cell differentiation. The degradation of phosphatidylcholine by phospholipases releases lysophosphatidylcholine (Zelenka et al. 1982) and may eventually produce diacylglycerol, lysophosphatidic acid, and other lipid mediators that could activate protein kinase C or other downstream effectors. However, these possibilities have not been tested.

9.4.1. Fiber Cell Elongation

Elongation is the morphological hallmark of fiber cell differentiation. However, it has been difficult to study this process in vivo at more than a descriptive level. Therefore, most mechanistic studies have used epithelial explants.

Chicken embryo lens epithelial cells double in length during the first five hours of culture in medium supplemented with vitreous humor (Beebe et al., 1980), insulin (Piatigorsky, 1973; Piatigorsky et al., 1973b), IGF-1 (Beebe et al., 1987), or fetal calf serum (Philpott and Coulombre, 1965; Piatigorsky et al., 1972). After this initial burst, elongation continues at a slower rate for weeks (Piatigorsky et al., 1973b). The rapid initial phase of elongation can occur in the absence of protein synthesis (Piatigorsky et al., 1972).

Colchicine, a drug that depolymerizes microtubules, inhibits the initial phase of elongation, suggesting that microtubules play an essential role in this cell shape change (Piatigorsky et al., 1972). However, nocodazole, another microtubule-disrupting drug, depolymerizes microtubules without preventing lens cell elongation in epithelial explants (Beebe et al., 1979). Therefore, intact microtubules are not essential for fiber cell elongation. Additional studies have demonstrated that colchicine inhibits lens cell elongation by a mechanism distinct from its ability to depolymerize microtubules (Beebe et al., 1979). The cell components that interact with colchicine to prevent elongation are not known.

As lens fiber cells elongate, in vivo or in vitro, they maintain a similar cross-sectional area. This means that their volume increases in proportion to their length, a phenomenon that has been confirmed by three-dimensional reconstruction of elongating lens cells in tissue sections (Beebe et al., 1982). Cell elongation could, therefore, be due to an increase in cell length that is precisely accompanied by an increase in cell volume or to an increase in volume that provides the driving force for the increase in cell length. Measurements of ion fluxes in elongating lens cells provide support for the latter explanation.

Stimulation of cell elongation leads to a change in the flux of potassium ions across the plasma membrane (Beebe et al., 1986; Parmelee and Beebe, 1988). Potassium continues to enter the cell at the same rate as before stimulation by the action of the Na,K-ATPase, but the potassium efflux slows, leading to the accumulation of potassium and chloride ions in the cytoplasm (Fig. 9.2). The resulting increase in the osmolarity of the cytoplasm leads to an influx of water and a concomitant increase in cell volume (Parmelee and Beebe, 1988; Beebe et al., 1990). In support of these data, agents that increase the efflux of potassium or reduce potassium uptake prevent cell elongation (Parmelee and Beebe, 1988; Beebe and Cerrelli, 1989).

Figure 9.2. Lens cell elongation is driven by the accumulation of potassium ions.

The cells at the lens equator or in an epithelial explant are all increasing in volume at the same time. They are also attached to their neighbors by cell-cell adhesive junctions and to the lens capsule by basal adherens junctions (Bassnett et al., 1999). Therefore, the increase in cell volume triggered by fiber differentiation factors is translated into an increase in cell length (Fig. 9.3; Beebe et al., 1986).

While this hypothesis has not been tested explicitly, isolated chicken embryo lens epithelial cells do not elongate when cultured in fetal bovine serum or vitreous humor. Similarly, at early stages of elongation, cells from epithelial explants round up when isolated from their neighbors (unpublished observations of D. Beebe). Therefore, it seems plausible that a physiologically regulated increase in cell volume and the constraints imposed by cell-cell and cell-substrate adhesions could be sufficient to account for the morphological changes that characterize the early stages of lens fiber cell differentiation. Continued elongation is also associated with a proportional increase in cell volume. This volume increase is likely to be driven by protein accumulation.

9.4.2. Increased Protein Synthesis Early in Fiber Cell Differentiation

In chicken embryo lens epithelial cells stimulated to elongate with fetal bovine serum, the absolute rate of protein synthesis (molecules/second/cell) increases nearly twofold in the first 5 hours (Milstone and Piatigorsky, 1975; Beebe and Piatigorsky, 1976). During this period, the rate of synthesis of the major lens crystallin in the embryonic lens, δ-crystallin, increases by a similar amount. In the next 19 hours of culture, total protein synthesis continues at about the same rate as seen at 5 hours, but the rate of δ-crystallin synthesis nearly doubles (Milstone and Piatigorsky, 1975; Beebe and Piatigorsky, 1976). The specific increase in δ-crystallin synthesis is associated with an increase in the accumulation of δ-crystallin mRNA (Milstone et al., 1976). There is no evidence of an increase in the

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Figure 9.3. Effect of volume increase on single cells or cells in an epithelial monolayer.

rate of protein degradation during this period, suggesting that the increased rate of protein synthesis is translated into increases in protein accumulation (Milstone and Piatigorsky, 1975; Beebe and Piatigorsky, 1976). Similar increases in total protein synthesis are likely to occur early in the differentiation of rodent lens epithelial explants, although analyses have not been performed as explicitly as in chicken lens explants. The increase in the size of nucleoli reported by Walton and McAvoy (1984) 16 hours after exposure of rat lens epithelial explants to retina-conditioned medium suggests a general increase in protein synthesis. By 24 hours after exposure to FGF, α-crystallin begins to accumulate (Peek et al., 1992). Therefore, in these in vitro model systems, fiber cell differentiation appears to be first associated with an overall increase in protein synthesis and accumulation, then with specialization for the synthesis and accumulation of the crystallins.

9.4.3. Protein Synthesis and Accumulation in Later Stages of Fiber Cell Elongation

The accumulation of crystallin and noncrystallin polypeptides during lens fiber cell differentiation may be responsible for the continued elongation of lens fiber cells. Inhibition of protein synthesis does not prevent the initial doubling of cell length in chicken embryo explants, but blocks subsequent elongation (Piatigorsky et al., 1972). Recent studies in which crystallin gene expression was inhibited in mice by the targeted deletion of essential transcription factors also provide evidence that protein accumulation drives

fiber cell elongation. Disruption of the transcription factor c-maf greatly reduces crystallin gene expression and fiber cell elongation (Kawauchi et al., 1999; Kim et al., 1999; Ring et al., 2000). Targeted deletion of the Sox1 or Prox1 transcription factors blocks fiber cell elongation at an early stage and prevents the accumulation of mRNAs for some of the γ -crystallins but not the α- and β-crystallins (Nishiguchi et al., 1998; Wigle et al., 1999). It is possible that the small amount of cell elongation that does occur when transcription factor genes have been knocked out reflects a low residual level of crystallin protein accumulation. Unfortunately, the amount of protein that accumulates in the lens cells of these genetically modified animals and the volumes of their shorter fiber cells have not been measured. Therefore, although these results are consistent with the idea that protein accumulation drives the later stages of lens fiber cell elongation, further study is needed to test this possibility.

It is interesting that the phenotype of the sox1 and prox1 knockout mice resembles that of the dominant Cat2elo mutation, which results in truncation of γ E-crystallin (Cartier et al., 1992). Apparently, normal expression of the γ -crystallin genes is essential for proper fiber cell elongation. It is not clear whether the inhibition of cell elongation that occurs when γ - crystallin synthesis is perturbed is due to a reduction in total protein accumulation or whether elongation depends in some specific way on having the proper complement of γ -crystallins.

The idea that fiber cell elongation is driven by an increase in cell volume is probably too simplistic to account for all aspects of fiber cell morphogenesis, but we know of no alternative mechanism that explains the coordinated increase in fiber cell length and volume. The lens fiber cell cytoskeleton may also contribute to the stabilization of the plasma membrane and to the development of the membrane specializations that are found in mature fiber cells (see below). However, the action of the cytoskeleton alone does not appear to be sufficient to account for fiber cell elongation.

Fiber cell elongation ceases when the apical and basal ends of the cells reach the sutures. Whether protein accumulation also slows or stops at this stage has not been determined. This is a critical stage in the life of a fiber cell, since suture formation involves separation of the fiber cell from the posterior capsule, establishment of connections with a fiber cell from the opposite hemisphere of the lens, and eventual burial of cells beneath layers of younger fibers. Little is known about changes in the synthetic or metabolic activity of fiber cells during this process.

9.4.4. Establishment of Regular Packing Order

As epithelial cells leave the transitional zone and begin to form fiber cells, a remarkable reorganization takes place. Instead of being randomly distributed within the cell layer, the early fiber cells line up in rows, or radial cell columns. This organization can be appreciated in whole mount preparations of the epithelium, where the aligned cells are referred to as “meridional rows” (see chap. 11). The precise alignment established in the meridional rows is maintained during the remainder of fiber cell differentiation.

Cross sections through epithelial cells show that the cell borders are irregular and that cells have variable numbers of neighbors. By contrast, fiber cells align with six neighboring cells in a precise packing geometry to form radial cell columns (Fig. 9.4). The cells in adjacent columns are interdigitated so that the cell nuclei in adjacent elongating fiber cells are displaced from each other by exactly one-half a cell thickness. Each fiber cell adheres to two fiber cells in the same radial column, one that is deeper in the lens and one that is more superficial. On each lateral border a fiber cell interacts with two cells in the adjacent radial cell column. This arrangement assures that fiber cells will have hexagonal cross sections.

Figure 9.4. The tissue organization of the embryonic chicken lens viewed perpendicular (A) and parallel to the optic axis (B).