Shortly after joining the brand new University of Washington Department of Ophthalmology in 1966, I discovered the classic book on human retinal development by Ida Mann. When a friend in the UW Primate Center asked me if I wanted the eyes from 16 timed fetal and infant M. nemestrina, I had my first opportunity to discover for myself the very strange development of the fovea. This talk will review the evidence that I and my collaborators have amassed on foveal development, and will present an integrated model of primate foveal development based on these data. Classic paper: Yuodelis, C., & Hendrickson, A. E. (1986). A qualitative and quantitative analysis of the human fovea during development. Vision Research, 26, 847–855.

The fovea centralis is a specialization of the primate retina to support high-acuity vision. It contains the highest density of red and green cone photoreceptors in the retina. Each cone has a unique private line synaptic circuit to midget ganglion cells which then transmit centrally a faithful representation of the high-density foveal cone mosaic. Blue cones and rods are absent from the human foveal center. Inner retinal layers are displaced peripherally during fetal development to form a pit which provides a stable optical path.

OBJECTIVE:To study the development of the perifoveal retinal vasculature.METHODS:We studied 7 retinas aged between 26 weeks' gestation and 1 week postnatal (41 weeks' gestation). Sections were imaged using high-resolution digital photography and blood vessel profiles identified at 200% to 300% magnification. Flat mounts were immunolabeled using antibodies to CD31 and factor VIII to identify blood vessels and antibodies to rhodopsin to identify the rod-free zone.RESULTS:The foveal region was identified by the absence of rod photoreceptors in the outer retina and/or presence of a shallow depression in the inner retina. The whole mount at 26 weeks' gestation showed a blood vessel-free region centered on the rod-free zone that was open along the horizontal meridian on the temporal side. At 37 weeks' gestation, the foveal avascular zone formed a complete circle. In sections, the foveal avascular zone was approximately 500 microm in diameter at 35 weeks' gestation and 300 to 350 microm at 40 weeks' gestation; in whole mounts, it was 150 to 170 microm in diameter at 37 and 41 weeks' gestation.CONCLUSIONS:The foveal region is normally avascular during development, as in adult life. We found no evidence of foveal vascularization during development of the human retina. Clinical Relevance Instances of vascularization of the foveal region are not due to failed regression of a transient vasculature.

By establishing an avascular, highly elastic, region within the fetal area of high acuity (AHA), the developing primate eye has created a unique substrate on which the mechanical forces of intraocular pressure (IOP) and growth-induced retinal stretch (stretch) can act. We proposed (Springer & Hendrickson, 2004b) that these forces generate both the pit and high cone density found in the adult AHA. In this paper, we use quantitative measures to determine the temporal relationships between nasal and temporal retinal elongation, changes in pit depth, cone packing, and cone morphology over M. nemestrina retinal development. Retinal length increased rapidly to about 105 days postconception (dpc; Phase 1) and then elongation virtually ceased (Phase 2) until just after birth (180 dpc). Retinal elongation due to stretch resumed during Phase 3 until approximately 315 dpc (4-5 months), after which time the retina appeared mature (Phase 4). The pit appeared during the quiescent Phase 2, suggesting that IOP acts, in conjunction with molecular changes in the inner retina, on the highly elastic, avascular, AHA to generate a deep, narrow pit and causes inner retinal cellular displacements. Subsequently (Phase 3), the pit widened, became 50% shallower and central inner retinal lamina thinned slightly due to a small amount of retinal stretch occurring in the AHA. Centripetal movement of cones was minimal until just after birth when the pit reached 88% of its maximal depth. Accelerated cone packing during Phase 3 was temporally correlated with increased stretch.

Cones in the foveola of adult primate retina are narrower and more elongated than cones on the foveal rim, which in turn, are narrower and more elongated than those located more eccentric. This gradient of cone morphology is directly correlated with cone density and acuity. Here we investigate the hypothesis that fibroblast growth factor (FGF) signaling mediates the morphological differentiation of foveal cones—in particular, the mechanism regulating the elongation of foveal cones. We used immunoreactivity to FGF receptor (R) 4, and quantitative analysis to study cone elongation on the horizontal meridian of macaque retinae, aged between foetal day (Fd) 95 and 2.5 years postnatal (P 2.5y). We also used in situ hybridization and immunohistochemistry to investigate the expression patterns of FGF2 and FGFR1–4 at the developing fovea, and three other sample locations on the horizontal meridian. Labeled RNA was detected using the fluorescent marker “Fast Red” (Roche) and levels of expression in cone inner segments and in the ganglion cell layer (GCL) were compared using confocal microscopy, optical densitometry, and tested for statistical significance. Our results show that morphological differentiation of cones begins near the optic disc around Fd 95, progressing toward the developing fovea up until birth, approximately. Levels of FGF2 and FGFR4 mRNAs expression are low in foveal cones, compared with cones closer to the optic disc, during this period. There is no similar gradient of FGF2 mRNA expression in the ganglion cell layer of the same sections. Maturation of foveal cones is delayed until the postnatal period. The results suggest that a wave of cone differentiation spreads from the disc region toward the developing fovea during the second half of gestation in the macaque. A gradient of expression of FGFR4 and FGF2 associated with the wave of differentiation suggests that FGF signalling mediates cone narrowing and elongation.

Most primate retinas have an area dedicated for high visual acuity called the fovea centralis. Little is known about specific mechanisms that drive development of this complex central retinal specialization. The primate area of high acuity (AHA) is characterized by the presence of a pit that displaces the inner retinal layers. Virtual engineering models were analyzed with finite element analysis (FEA) to identify mechanical mechanisms potentially critical for pit formation. Our hypothesis is that the pit emerges within the AHA because it contains an avascular zone (AZ). The absence of blood vessels makes the tissue within the AZ more elastic and malleable than the surrounding vascularized retina. Models evaluated the contribution to pit formation of varying elasticity ratios between the AZ and surrounding retina, AZ shape, and width. The separate and interactive effects of two mechanical variables, intraocular pressure (IOP) and ocular growth-induced retinal stretch, on pit formation were also evaluated. Either stretch or IOP alone produced a pit when applied to a FEA model having a highly elastic AZ surrounded by a less elastic region. Pit depth and width increased when the elasticity ratio increased, but a pit could not be generated in models lacking differential elasticity. IOP alone produced a deeper pit than did stretch alone and the deepest pit resulted from the combined effects of IOP and stretch. These models predict that the pit in the AHA is formed because an absence of vasculature makes the inner retinal tissue of the AZ very deformable. Once a differential elasticity gradient is established, pit formation can be driven by either IOP or ocular growth-induced retinal stretch.

We analysed spatial density and distribution of short-wavelength-sensitive photoreceptors (S-cones) in developing and adult human retinae using antibody against short-wavelength-sensitive opsin. Statistical tests indicate that before 20 weeks of gestation (WG) the S-cone mosaic is not distinguishable from a random distribution, but by 20 WG is significantly different from a random distribution in the perifoveal region, as reported previously for adult retina. Changes in spatial density during development are consistent with displacement of the photoreceptor population towards the incipient fovea so that prior to 20 WG, peak S-cone density is >1.7 mm from the fovea, but is within 800 μm of the fovea by 20 WG.

Mechanisms underlying the development of the primate area of high acuity (AHA) remain poorly understood. Finite-element models have identified retinal stretch and intraocular pressure (IOP) as possible mechanical forces that can form a pit (Springer & Hendrickson, 2004). A series of Macaca nemestrina monkey retinas between 68 days postconception (dpc) and adult were used to quantify growth and morphological changes. Retinal and pars plana length, optic disc diameter, disc-pit distance, and inner and outer retinal laminar thickness were measured over development to identify when and where IOP or stretch might operate. Horizontal optic disc diameter increased 500 mum between 115 dpc and 2 months after birth when it reached adult diameter. Disc growth mainly influences the immediate surrounding retina, presumably displacing retinal tissue centrifugally. Pars plana elongation also began at 115 dpc and continued steadily to 3-4 years postnatal, so its influence would be relatively constant over retinal development. Unexpectedly, horizontal retinal length showed nonlinear growth, divided into distinct phases. Retinal length increased rapidly until 115 dpc and then remained unchanged (quiescent phase) between 115-180 dpc. After birth, the retina grew rapidly for 3 months and then very slowly into adulthood. The onset of pit development overlapped the late fetal quiescent phase, suggesting that the major mechanical factor initiating pit formation is IOP, not retinal growth-induced stretch. Developmental changes in the thickness of retinal layers were different for inner and outer retina at many, but not all, of the ten eccentricities examined.

In primates, short wavelength sensitive cones (S cones) and medium- or long-wavelength-sensitive cones (L/M cones) are two separate populations. Each cone type has a different developmental timecourse, contributes to different intra-retinal circuits, and transmits different types of information to the brain. However, in fetal human retina a significant population of cones express both S and L/M opsin (S + L/M cones), raising questions about whether S + L/M cones die or change opsin expression during development. We have utilized fetal, postnatal and adult human retinae to study the immunohistochemical distribution and morphology of S + L/M cones during development. Because S cones appear to be at higher density in fetal compared to adult retinae, we used antibodies to S opsin and alpha-transducin to estimate the proportion of S-cones, and TUNEL labelling to detect apoptotic death in the L/M, S or S + L/M population during development.S cones were present in central retina from fetal week (Fwk)11 and covered the retina by Fwk20. L/M cones appeared in the foveal cone mosaic 3-4 weeks after S-opsin was first detected, and covered the retina by birth. S + L/M cones were detected in all retinae older than Fwk14. They were most numerous at the retinal eccentricity where L/M opsin was just appearing; i.e. at the 'front' of L/M opsin expression. In this region, five morphological types of cones were present. (1) Heavily labelled S cones had thick cell bodies, a thick basal axon and pedicle, and a nucleus at any level of the outer nuclear layer (ONL). (2) Heavily labelled L/M cones were wine goblet shaped with a small round cell body, a large nucleus at the outer ONL edge, and a thin axon with a prominent synaptic pedicle. (3) Goblet-shaped S + L/M cones. (4) Goblet-shaped cones lightly labelled for S-opsin. (5) Cones that were not immunoreactive to either opsin. Only type 1 S cones were present peripheral to the L/M expression front, and their labelling intensity, morphology and distribution indicates that these are the 'true blue' cones of the adult mosaic. Only type 2 L/M cones were present in the foveal cone mosaic. Types 3 and 4 were most numerous within 500-750 mum of the L/M expression front, but type 3 S + L/M cones were also scattered throughout more central regions in fetal, infant and adult retinae. S + L/M cones comprised 5-10% of opsin immunoreactive cones at the L/M front in fetal and early postnatal retinas but 0(.)01-0(.)03% throughout P8mo and adult retinae. We found no evidence of significant levels of apoptosis in L/M cones at the expression front, suggesting that this decrease was not due to cell death. The findings suggest that goblet-shaped cones destined to express L or M opsin may initially and transiently express S opsin.Near the optic disc, at Fwk17 S cone density was around 2000 cells mm(-2), which dropped 50% by Fwk20 and stabilized at around 500 cells mm(-2) by birth. Double labelling with alpha-transducin showed that throughout this period 8-10% of all cones expressed S opsin. TUNEL labelling found no significant apoptosis in the S cone population. The decrease in S cone density near the optic disc occurs in the absence of apoptosis, and is likely due to other developmental events acting on the photoreceptor layer, including displacement of cones towards the fovea. (C) 2004 Elsevier Ltd. All rights reserved.

In macaque monkeys the foveal depression forms between fetal day (Fd) 105 and birth (Fd 172 of gestation). Before this, the incipient fovea is identified by a photoreceptor layer comprising cones almost exclusively, a multilayered ganglion cell layer (GCL), and a “domed” profile. Vessels are absent from the central retina until late in development, leading to the suggestion that the GCL in the incipient fovea may be transitorily hypoxic. Vascular endothelial growth factor (VEGF), expressed by both glial and neuronal cells and mediated by the hypoxia‐inducible transcription factor (HIF)‐1, is the principal factor involved in blood vessel growth in the retina. We examined VEGF expression in macaque retinas between Fd 85 and 4 months postnatal. Digoxygenin‐labeled riboprobes were generated from a partial‐length human cDNA polymerase chain reaction fragment, detected using fluorescence confocal microscopy, and quantified using Scion Image. High levels of VEGF mRNA were detected in astrocytes associated with developing vessels. We also detected strong expression of VEGF mRNA in the GCL at the incipient fovea prior to Fd 105, with peak labeling in the incipient fovea that declined with distance in nasal and temporal directions. By Fd 152 peak labeling was in two bands associated with development of the inner nuclear layer (INL) capillary plexus: in the inner INL where Müller and amacrine cell somas are located, and in the outer INL where horizontal cells are found. The findings suggest that at the incipient fovea the GCL is hypoxic, supporting the hypothesis that the adaptive significance of the fovea centralis is in ensuring adequate oxygen supply to neuronal elements initially located within the avascular region. J. Comp. Neurol. 462:42–54, 2003. © 2003 Wiley‐Liss, Inc.

PURPOSE:To better characterize the cellular constituents of cysts in the peripheral retina and pars plana of the adult monkey.METHODS:Frozen sections of the peripheral retinal margin and pars plana from monkeys (Macaca nemestrina) between 1 and 15 years of age were stained with toluidine blue or immunolabeled with a variety of glia- and neuron-specific antibodies.RESULTS:In animals 1 to 2 years of age, the nonpigmented inner layer of the pars plana is a pseudostratified columnar epithelium. In these young animals, the peripheral retina had distinct layers and did not contain cysts. In animals 6 years of age or older, there were numerous cysts in the pars plana and in the peripheral retina. In the peripheral retina, neurons were randomly distributed and did not have a laminar organization. Cells surrounding cysts were immunoreactive for different types of markers for retinal neurons. Some of the cells surrounding cysts in the pars plana were also unexpectedly immunoreactive for antigens normally expressed only in retinal neurons and glia.CONCLUSIONS:Cysts form in the peripheral retina and pars plana in adult monkeys. The peripheral retinal cysts disrupt the normal lamination of the cells, but all types of retinal neurons are still present in the cysts. In an unexpected finding, cysts in the pars plana also contained cells immunoreactive for a few of the markers of retinal cells, suggesting that neurogenesis may occur in the pars plana of the adult primate.