Ординатура / Офтальмология / Английские материалы / Retinal Development_Sernagor, Eglen, Harris, Wong_2006

Numerous processes cluster below the cones in the narrow OPL, but it is difficult to identify their origin at this age. Synaptic vesicle protein labelling in cones spreads across the retina, reaching the optic disc at Fd 85 to 90 and the retinal edge by Fd 125. Thus, cone synaptic formation is initiated even before a distinct OPL can be identified. Although primate rod synaptogenesis has not been reported in detail, rod synapses appear to form later than those of cones at every point on the retina (Hollenberg and Spira, 1973; Okada et al., 1994). The sequence of OPL synaptic types has not been determined for primates, but in other mammals HC processes seem to make the first contact with ribbon synapses (reviewed in Hendrickson (1996)). In monkeys there is a relatively long delay between cone and HC genesis at Fd 36 and synaptic formation at Fd 60. This suggests that synaptic formation is delayed until BC dendrites are present and that, even if HC processes form the earliest contacts, they are not sufficient to initiate synaptogenesis.

7.3.3 The general pattern of expression of phototransduction proteins

Although Macaca monkey and human cone and rod photoreceptors seem to be identical in morphology, electrophysiology and biochemistry (Nathans, 1989; Jacobs, 1996; Jacobs and Deegan, 1999), their pattern of opsin expression is not. Riboprobes and antisera have been produced that are specific for S- vs. L-/M-opsin, but none are available that can differentiate L- from M-opsin; therefore the term L-/M- will be used to refer to the red and green opsins (see Bumsted et al., 1997, for discussion). In humans (Xiao and Hendrickson, 2000) the first opsin to appear is in S-cones and can be detected around the fovea at Fwk 12 (Figure 7.4a). In both cones and rods, when opsin is expressed the photoreceptor has formed a short outer segment (Dorn et al., 1995). L-/M-opsin is not detected until Fwk 15 in the foveal centre. S-opsin expression spreads rapidly across the retina so that S-cones are present at all retinal edges by Fwk 22, whereas L-/M-opsin expression is only at the eccentricity of the OD at Fwk 20 and does not reach the retinal edge until near birth. Rod opsin expression in humans follows that of L-/M-opsin, appearing on the foveal edge at Fwk 15 and reaching the retinal edge before birth. By contrast, in Macaca monkeys (Bumsted et al., 1997) both S- and L-/M-opsin appear within the foveal region at Fd 65 to 70 (Figure 7.4b). S-opsin expression then spreads rapidly across the retina, reaching the retinal edge by Fd 135 while L-/M-opsin is not present at the edge until Fd 150. Rod opsin also appears around the monkey fovea at Fd 65 to 70 and spreads across the retina to reach its edge by Fd 135 (Dorn et al., 1995).

Comparison of Figures 7.4a and 7.4b suggests a similar pattern for L-/M-opsin in both humans and Macaca monkeys, but widely different expression patterns for S- and rod opsin in the two species. Another major difference between species is that there is a transient expression of S-opsin at the onset of L-/M-opsin in some human cones producing transient ‘S- + L-/M-’ cones that have not been detected in macaques (Cornish et al., 2004b). S-opsin disappears with age in almost all L-/M-cones but this is not associated with cell death; in fact, cell death among the cone population at any age is very low (Georges et al., 1999; Cornish et al., 2004b). Transient expression of S-opsin may in some way direct the future choice of L- or M-opsin. Because a few cones expressing both S- and L-/M-opsins remain throughout all adult human retinas examined, coexpression is compatible with continued

survival and presumed function, similar to other mammalian retinas where this occurs (see Cornish et al., 2004b). In both primates, outer-segment length increases for many months after birth. For instance, mid-peripheral rod outer-segment length increases 50% between Fd 115 and birth, and a further 50% between birth and adulthood (Dorn et al., 1995). Foveal cone outer-segment length in humans increases from 3 µm at birth to 50 to 60 µm in the adult (Yuodelis and Hendrickson, 1986).

Expression of proteins involved in the phototransduction cascade has only been followed in Macaca monkey cones (Sears et al., 2000). Structural proteins of the outer segment like peripherin appear simultaneously with opsin and morphological optic stalk (OS) in both S- and L-/M-cones. Phototransduction proteins such as cone-specific alpha-transducin, phosphodiesterase and rhodopsin kinase appear shortly after L-/M-opsin expression. Curiously, although S-cones express opsin earlier than L-/M-cones, expression of phototransduction proteins is delayed in S-cones until surrounding L-/M-cones are immunoreactive for both opsin and these proteins.

7.4The fovea

7.4.1Formation of the foveal depression

Pre-pit stage

The incipient fovea can be identified temporal to the OD by Fwk 11 in humans and Fd 50 to 55 in macaques, but up to midgestation this region remains relatively unchanged morphologically (Figures 7.3 and 7.5). The incipient fovea is characterized by a single layer of cones in the ONL (Figure 7.5A–D) and has been termed the rod-free zone (Hendrickson and Kupfer 1976) because rods are virtually absent from its centre. It also has been called the pure-cone area for similar reasons (Springer and Hendrickson, 2004). To emphasize its continuity with the cone mosaic across the retina, it has also been termed the foveal cone mosaic (Provis et al., 1998; Cornish et al., 2004a). Here we use the term foveal cone mosaic because the fetal ONL, even within the incipient fovea, includes scattered rods. The remaining layers are a thin OPL and thick INL, IPL and GCL. In fact, the early foveal GCL is the thickest in the retina and often bulges into the vitreous so that it can be detected histologically. Another characteristic is that the eye is oval shaped at these stages with a distinct bulge temporal to the OD (not shown), which includes the incipient fovea. In this region the retinal pigment epithelium (RPE) is more pigmented than in the periphery and the retina seems to be tightly attached to the outer eye layers.

A recent study comparing expression patterns as detected by RT-PCR and immunocytochemistry (Bumsted-O’Brien et al., 2003) finds that immunolabelling is more sensitive for detecting small numbers of cells expressing markers early in development of the fovea. By both methods photoreceptor proteins such as phosphodiesterase, CRX, interphotoreceptor retinoid-binding protein, Nrl and S-opsin are expressed at Fwk 9 to 12. All first appear in foveal cones or rods at the foveal edge. L-/M-opsin and rod opsin were detected at Fwk 15 to 16, consistent with immunolabelling (Xiao and Hendrickson 2000). Immunolabelling

of synaptic proteins also finds that foveal cones express synaptophysin, SV2, and glutamate vesicular transporter as well as markers for synaptic ribbons by Fwk 11. Electron microscopy studies find SV2 labelling and synaptic ribbons and vesicles in monkey cones by Fd 60 (Okada et al., 1994) while neither S- or L-/M-opsin is detected until Fd 70 (Bumsted et al., 1997). Thus, one distinctive pattern of putative L-/M-cones is that they form synapses very early, and at least a month before they express opsin. S-cones in humans seem to form synapses shortly before they express opsin.

Inner neurons also mature rapidly. As described above (p. 131), IPL synaptogenesis in the fovea is well advanced by Fd 85 in monkeys. Immunolabelling finds that characteristic markers for BCs such as Goα for ON-BCs, recoverin for OFF-BCs and protein kinase α for rod-BCs as well as glutamate receptors 2/3 and 5 are all present in the Fwk 12 to 14 INL, OPL and IPL (Hendrickson, unpublished data). Amacrine cell markers γ- aminobutyric acid (GABA) and glycine also are present in the IPL although adult banding patterns are not yet clear. Thus, by midgestation, at Fwk 20 in humans and Fd 85 in monkeys, although the incipient fovea has not changed much morphologically, its neurons are forming their characteristic synaptic circuits and have expressed phototransduction proteins.

Pit stage

The superior and inferior vascular arcades (Provis et al., 2000; Provis, 2001) grow from the OD to vascularize the central retina. These arcades grow around the fovea, never entering the central region. Rather, they form instead the FAZ (Gariano et al., 1994; Provis et al., 2000). The FAZ remains stable at approximately the diameter of the future pit rim, about 500 µm. The first sign of pit formation is a slight thinning of the GCL at Fwk 25 to 26 in humans (Figure 7.5E–H) and Fd 105 to 110 in monkeys (Figure 7.2C–F). This thinning occurs within the FAZ and is approximately centred on the foveal cone mosaic. The pit forms by a sequential indentation of GCL, IPL, INL and finally the OPL.

Two important points should be emphasized about pit formation. The first is that inner retinal neurons and M¨uller glia are displaced toward the periphery (centrifugally); they do not die by apoptotic cell death (Georges et al., 1999). The second is that this displacement takes place after synapses have been formed in both the IPL and OPL so that the mechanisms causing this displacement must be able to retain these synaptic connections.

The foveal pit is still relatively immature at birth in humans (Figure 7.5G) with thin IPL and GCL layers and a thick INL. The ONL is still a single layer of elongated cones that tilt toward the centre of the pit, but are not yet tightly packed. On the other hand, the neonatal macaque fovea is relatively much more mature. By Fd 130 (Figure 7.2C) the fovea resembles the neonatal human fovea and by birth (at Fd 174) there is a well-formed pit with a single layer of cells across the bottom (cf. Figure 7.2d). Neonatal monkey cones also have begun to show evidence of packing with a significant elongation of cone cell bodies and cone nuclei packed one to two deep especially on the rim.

7.4.2 Cone packing

Quantitative studies show that both rods and cones are displaced toward the foveal centre (centripetally) during the pre-fovea stage (Diaz-Araya and Provis, 1992). When they first differentiate, cones in the foveal cone mosaic have a packing density of approximately 12 000/mm2 (Figure 7.6a). By Fwk 30 cone density at the fovea is around 30 000/mm2, with no addition of new cells (Yuodelis and Hendrickson, 1986; LaVail et al., 1991). This increase is accompanied by a decrease in size of the foveal cone mosaic diameter prenatally – suggesting that cones and rods are displaced towards the pit centre (Hendrickson and Kupfer, 1976; Youdelis and Hendrickson, 1986; Diaz-Araya and Provis, 1992). Analysis of the S-cone population near the OD of human retina shows that photoreceptors even at this relatively peripheral location are displaced towards the foveal cone mosaic between Fwk 17 and 6 weeks postnatal (Cornish et al., 2004a). This emphasizes the protracted period and the large retinal area involved in cone displacement.

A more rapid phase of cone packing begins around birth. Based on thickness of the ONL, this occurs in humans and macaques, but detailed quantitative data comes from study of the macaque retina (Packer et al, 1990; Springer & Hendrickson, 2005). Cone density rises from 65 000/mm2 at 3 weeks to 140 000/mm2 by one year and will eventually reach 200 000/mm2 by 1.5 years of age which is equal to a 6 year human. Humans have an even longer developmental time period, with neonatal densities around 36 000/mm2 and the lowest range of adult density reached at 45 months postnatal (Yuodelis and Hendrickson, 1986), although precisely when final adult density is reached is not certain.

This marked increase in cone density is reflected in a striking change in cone morphology (Hendrickson & Kupfer, 1976; Yuodelis and Hendrickson, 1986; Diaz-Araya and Provis, 1992), shown diagrammatically in Figure 7.6b. Cones in the foveal cone mosaic at Fwk 22 are relatively simple cuboidal cells, 8 to 10 µm in diameter. By birth both an inner and outer segment are present as well as a distinct synaptic pedicle; the inner segment is 5 to 7.5 µm wide and 7.5 to 10 µm long, with a short outer segment 3 to 4 µm in length. By 12 to 15 months the elongated inner segment is half the neonatal width and almost 25 µm long. Adult cones are about 2 µm in diameter with an inner segment 30 to 35 µm long and an outer segment 50 to 65 µm long.

Another striking change in cone morphology involves the change in cone angle and the appearance of an axon or Henle fibre (Figures 7.6b, 7.7). Initially, the synaptic pedicle is located immediately adjacent to the nucleus, and there is only a short axon or Henle fibre (Hendrickson and Yuodelis, 1984). Next, the long axis of the cone tilts, so that the elongating inner segment points toward the centre of the pit and the Henle fibre extends laterally away from the centre of the pit. This change in morphology occurs as the INL neurons are displaced laterally (Figure 7.7). In monkeys this is in the late prenatal stage while in humans it occurs before and just after birth. Elongation of the axon appears to be a morphological adaptation of cones that enables retention of synaptic connections, made in the ‘pre-pit’ stage, with HCs and BCs. As BCs and HCs move out of the deepening pit,

Figure 7.6 Cones pack into the central fovea over a protracted period. This packing is associated with morphological adaptation of individual cones. (a) Cone density at the central fovea in human and macaques is plotted as a function of time, measured as a percentage of the caecal period – the time between conception and eye opening. The period during which the foveal pit forms is also included on the same axis. The graph shows that cone packing takes place over a protracted period, starting before the fovea begins to form and continuing for a considerable time afterwards. aCurio et al. (1990); bPerry and Cowey (1985), Packer et al. (1989), Wikler et al. (1990). (b) Morphological differentiation of cones takes place over the same time-frame, with much of the specialization occuring postnatally in humans.

(a)

BC

GC

(b)

(c)

BC

Figure 7.7 A diagrammatic summary of foveal development. (a) In the first stage (Fd 55 to 100 or Fwk 11 to 24) the fovea has five distinct layers. The ONL is a single layer of cones, which have a short Henle axon extending vertically to contact underlying BCs. Bipolar axons are also vertical in the IPL. Arrows indicate the presumed direction of cell displacement. (b) In the second stage (Fd 100 to P1 months or Fwk 24 to P4 to 6 months) the pit invaginates the inner retinal layers, displacing the neurons peripherally. This is reflected by a peripheral slant to the BC axons as GCs are displaced laterally. Note that this slant appears on cone FH on the foveal slope, suggesting this is where cone packing toward the pit centre is initiated. (c) During the final stage (P1 to 12 months or P6 months to 4 years) the pit becomes deeper, all inner retinal neurons are displaced peripherally and cones form a thick layer over the pit so that cone density rises 10×. Cone pedicles are displaced peripherally to retain their synaptic contact with the more peripheral BCs and HCs. For colour version, see Plate 7.