Fig. 1.16  Comparison of the expression of CRALBP in the adult and developing retina. The photomicrograph is from a 2.5-month- old albino rat retina probed with antibody directed against CRALBP. At this age, immunospecific reactivity for CRALBP is present not only in the RPE but also in the Müller cell glia (compare with Fig. 1.15). The Müller cells span the entire thickness of the neural retina; sclerad, their villous processes protrude into the subretinal space; vitread, the Müller foot processes form the inner limiting membrane, a true basement membrane lining the inner retinal surface. The inner limiting membrane does not take up the tolulidine counter stain well. Its location is designated by the slanted arrowhead. Insert shows the pattern of CRALBP expression in the developing retina at postnatal day 2. Mitotic divisions are present at the outer edge of the neuroblastic epithelium (white arrows). Note that although the RPE expresses CRALBP, at this age, no expression is present in developing neural retina. This differential expression of CRALBP in the embryonic retina compared to that in the adult retina is due to the fact that the RPE differentiates early during retinal development, while the Müller cells emerge relatively late (compare with the histogenesis birth order summary in Fig. 1.14). Black arrowhead, separation artifact of the neruoblastic epithelium from the apical RPE surface; White arrowheads, RPE nuclei; Black arrow, ganglion cell nucleus. The antibody used for this immunohistochemical study was provided by Dr. Jack Saari. The images represent unpublished data from the author’s laboratory

There appears to be a state of competence allowing cells to respond to environmental cues [291]. This competence is controlled by intrinsic cues and cannot be modified [292]. In contrast, how competent cells will respond depends on a combination of positive and negative cues (reviewed in [287, 293–295]). Insight

into the connection between birth order and competence comes from studies where the cell cycle is experimentally altered in the developing peripheral Xenopus retina. As shown in Fig. 1.18, the peripheral retina of the Xenopus is less differentiated­ than that of the central retina. In most vertebrates, retinal differentiation proceeds from a central-to-peripheral direction. Furthermore, in many animals, such as fish, frogs, and birds where the eye continues to enlarge significantly beyond the postnatal period, continued retinoblast proliferation allows the retina to expand together with the enlarging eye. The addition of mature retina is largely due to the proliferation of stem cells located in the peripheral retina. This proliferation with continual cell cycle exiting of some of the progenitor daughters provides a wonderful system to study the relationship between external cues and cellular competence in the control of cell fate [296]. Ohnuma et al (2002). took advantage of this feature in the developing Xenopus retina to explore the coordination between birth order and the relative timing of cell cycle exit [54]. To control cell cycle exit, these investigators missexpressed specific cell kinase inhibitors. Using this strategy, they found that early cell cycle exit enhances the retinal ganglion cell promoting activity of Math5 [54, 297]. Furthermore, inhibiting cell cycle exit biases the cell toward later retinal neuron cell fates. Taken together, these data suggest a model of the histogenesis in which early cell cycle withdrawal enhances the activity of factors that promote early cellular fates. Conversely, late cell cycle withdrawal inhibits proneural function and pushes cells toward later fates. In summary, an interplay between cell cycle control and cellular determination helps to coordinate retinal histogenesis (see also [298–305]).

1.9  Focusing on the Fovea: A Marvel of Development

In view of the importance of the fovea to vision, it is remarkable that so little is known regarding the mechanism of its formation. The fact that the structure is virtually restricted to primates may be part of the problem. As a result, the techniques and the experimental approaches proven so powerful in systems such as Drosophila and mice may not be applicable to understanding histogenesis of a structure not

Fig. 1.17  Structure of the normal adult human retina (peripheral macula region). The retina is a highly ordered structure with its various cell types arranged in discrete lamina. The layers are typically named according to their relative position with respect to the vitreous. Layers closer to the center of the eye are termed “inner,” and those closer to the sclera as “outer.” Within each layer, various cell types can be identified. For example, cone cell nuclei line up on the outer edge of the ONL. The remainder of the nuclei in the ONL belongs to the rods (compare with higher magnification photomicrographs in Fig. 1.18). Note that the

domain from RPE through ONL is avascular. For detailed description of the anatomy of the human retina with wonderful photomicrographs and drawings, the reader is referred to Hogan’s atlas of the human eye [334]. Labels: Scl sclera; C choroid; RPE retinal pigment epithelium; BM Bruch’s membrane; OS outer segments; IS inner segment; ONL outer nuclear layer; INL inner nuclear layer; GCL ganglion cell layer. The section was stained with hematoxylin and eosin. Image from the author’s laboratory and Ophthalmic Pathology Service of the Ross Eye Institute

present in those animals. Nevertheless, many of the principles of retina development discussed above will certainly have central roles. For example, pax has a key function as mutations in this gene disrupt the normal development of the fovea [306–308]. A wealth of background information on its structure, function, and

development may be found in a number of thoughtful reviews [309–314].

The fovea is characterized by a high density of cone photoreceptors centered within its depression, a unique neuronal circuitry, and absence of a local retinal-blood supply. Remarkably, the location of the future fovea is

Fig. 1.18  Photomicrograph of the Xenopus laevis retina corresponding to the early free swimming tadpole stage. The photomicrograph compares the peripheral retina (asterisk) with the more central retina. Note the absence of photoreceptor differentiation peripherally. The central retina is more advanced showing definite outer and inner nuclear layers (ONL, INL) and photoreceptor differentiation with early outer segment formation (arrows). The retinal pigment epithelial (RPE) cells are laden with melanin granules. The final number and types of cells that comprise the adult vertebrate retina are determined not only by control of cellular proliferation but also through elimination of extra cells by programmed cell death. The bracket within the INL identifies a cell showing the typical nuclear fragmentation of apoptosis. Arrowhead, RPE nuclei; C cone nucleus; R rod nucleus. The specimen was embedded in plastic resin, and the section stained with toluidine blue; Unpublished data from the author’s laboratory

evident as early as 11 weeks gestational age in man [312, 315–317]. Figure 1.19 compares the fovea and midperipheral normal adult human retina in panels A and B, respectively. The panels, which are at identical magnifications, demonstrate the higher cone-packing density characteristic of the fovea made possible by the more slender shape of the foveal cones compared to those in other regions of the macula. Although the outer segments are longer in the fovea, the separation of the RPE from the neural retina in panel A is partly a technical artifact.

To maximize the resolving power of the fovea, the developing retina introduces striking local structural changes. The ganglion cells and the inner nuclear layer are eliminated from the fovea. The absence of these cells results in the foveal depression or pit, which is evident in second half of gestation and is incomplete at birth. The central region of the depression or foveola is 300–400 mm in diameter. The reduced number of noncone cells in the fovea may be due to increased removal of these cells and/or decreased production of the cells during foveal development. One mechanism to

eliminate noncone neurons in the fovea might be through programmed cell death. Cells undergoing apoptosis can be identified by on histological sections using terminal­ transferase dUTP-biotin nick-end labeling (TUNEL). However, studies of the rate of apoptosis in the ganglion cell and inner nuclear layers of human fetal retina between 14 and 35 weeks of gestation “indicate that the formation of the foveal depression cannot be attributed to death of cells in the ganglion cell layer and/or the inner nuclear layer” [318].

A more attractive idea is that the foveal depression forms not through apoptosis, but rather because cell bodies of the ganglion and inner nuclear layer cells move laterally relative to the foveal center. This displacement begins during late fetal life and continues through the early infant years. Thus, the available evidence, although largely descriptive in nature, is consistent with a model that the foveal depression forms as a consequence of centrifugal migration or displacement [317]. For example, the foveal cones, which made synaptic contacts to corresponding bipolar cells before the formation of the foveal depression, remain connected through the Fibers of Henle to their bipolar cells now located in the foveal rim of the adult retina. Furthermore, the displacement of the ganglion cell somata from their dendritic terminals in the foveal rim of the adult fetal monkey provides anatomical evidence that the ganglion cell somata have been displaced centrifugally from their bipolar cell contacts [319]. Although these anatomic observations suggest that the inner retinal neurons and glia are laterally displaced during development, it should be kept in mind, as pointed out in the detailed review of Provis et al. [317], that there is no direct experimental evidence for this model.

A further remarkable characteristic of the fovea is that it is an area entirely devoid of retinal vessels, including capillaries [320–322]. As will be discussed below, this feature may be linked to the mechanisms responsible for the formation of the foveal pit. The absence of retinal vessels in the fovea has the advantage of reducing optical interference. This strategy is not novel in the animal kingdom as the retinas of most avian species lack an inner vascular layer, a feature contributing to the high visual acuity of birds. In primates, the avascular zone is limited to the fovea. What accounts for the avascular zone, and how does it come to be centered over the fovea? Finally, do the limitations on vascularization of central retina at least in part account for its vulnerability to degenerative changes, as seen in age-related macular degeneration?

Fig. 1.19  Comparison of the foveal, and midperipheral normal adult human retina in (a, b) respectively. The photomicrographs, which are at identical magnifications, demonstrate the higher cone-packing density characteristic of the fovea. The higher density is made possible by the slender morphology of the foveal cones in this (compare with the cone cell widths in (b)). Although the outer segments are indeed longer in the fovea, the separation of the RPE from the neural retina in (a) is a tissue-processing artifact. The RPE shows a greater melanin density in the fovea. The ELM is not an actual membrane but only appears as one at low magnification. The apparent absence of the ELM in the fovea may represent an anatomic difference in this region [335]. At high magnification (insert), the ELM actually represents the junctional complexes (zonulae adherens) linking individual photoreceptors to adjacent Müller cells [336]. Although the Müller cells cannot be appreciated in the photomicrographs, close inspection of the insert shows two z. adherens complexes (arrows) that are

separated­ by Müller cell cytoplasm. In this location, villous processes extend from the Müller cell into the subretinal space, which is filled with an extracellular material known as the IPM. The IPM, which fills the subretinal space in both the foveal and nonfoveal retina, can be demonstrated by the use of special stains, immunohistochemical studies, and transmission electron microscopy (not illustrated). Labels: BM Bruch’s membrane; RPE retinal pigment epithelium; IS inner segment; ONL outer nuclear layer; OPL outer plexiform layer; INL inner nuclear layer. Labels within insert: COS cone outer segment; e ellipsoid (mitochondrial rich region); m myoid (zone between ellipsoid and nucleus containing much of the cell’s protein synthesis machinery including the endoplasmic reticulum, ribsomes, and golgi apparatus). The sections were stained with hematoxylin and eosin and photographed under 60× oil immersion. Images from the author’s laboratory and Ophthalmic Pathology Service of the Ross Eye Institute

Some clues to these questions may be found by considering the relationship of the fovea to the retinal vessels. The radial pattern of vessel growth around the incipient fovea has suggested the idea that there are factors expressed near the fovea that attract vessel growth. The absence of vessels in the central retina could be explained by the presence of inhibitory factors excluding vessels from the central region. In the developing monkey retina, where these concepts have

recently been examined, the fovea can be ­appreciated in the last trimester as a region consisting of cones and ganglion cells with an overall “domed” ­profile [323]. In this system, vessels are absent from the central retina until late in development. Vascular endothelial growth factor (VEGF), expressed by both glial and neuronal cells and mediated by the ­hypoxia-inducible transcription factor (HIF)-1, may be important in triggering blood vessel growth in the retina. These vessels are