Characterization of the development of the high-acuity area of the chick retina.

The fovea is a small region within the central retina that is responsible for our high acuity daylight vision. Chickens also have a high acuity area (HAA), and are one of the few species that enables studies of the mechanisms of HAA development, due to accessible embryonic tissue and methods to readily perturb gene expression. To enable such studies, we characterized the development of the chick HAA using single molecule fluorescent in situ hybridization (smFISH), along with more classical methods. We found that Fgf8 provides a molecular marker for the HAA throughout development and into adult stages, allowing studies of the cellular composition of this area over time. The radial dimension of the ganglion cell layer (GCL) was seen to be the greatest at the HAA throughout development, beginning during the period of neurogenesis, suggesting that genesis, rather than cell death, creates a higher level of retinal ganglion cells (RGCs) in this area. In contrast, the HAA acquired its characteristic high density of cone photoreceptors post-hatching, which is well after the period of neurogenesis. We also confirmed that rod photoreceptors are not present in the HAA. Analyses of cell death in the developing photoreceptor layer, where rods would reside, did not show apoptotic cells, suggesting that lack of genesis, rather than death, created the "rod-free zone" (RFZ). Quantification of each cone photoreceptor subtype showed an ordered mosaic of most cone subtypes. The changes in cellular densities and cell subtypes between the developing and mature HAA provide some answers to the overarching strategy used by the retina to create this area and provide a framework for future studies of the mechanisms underlying its formation.

Development of cone photoreceptors and their synapses in the human and monkey fovea.

During retinal development, ribbon synapse assembly in the photoreceptors is a crucial step involving numerous molecules. While the developmental sequence of plexiform layers in human retina has been characterized, the molecular steps of synaptogenesis remain largely unknown. In the present study, we focused on the central rod-free region of primate retina, the fovea, to specifically investigate the development of cone photoreceptor ribbon synapses. Immunocytochemistry and electron microscopy were utilized to track the expression of photoreceptor transduction proteins and ribbon and synaptic markers in fetal human and Macaca retina. Although the inner plexiform layer appears earlier than the outer plexiform layer, synaptic proteins, and ribbons are first reliably recognized in cone pedicles. Markers first appear at fetal week 9. Both short (S) and medium/long (M/L) wavelength-selective cones express synaptic markers in the same temporal sequence; this is independent of opsin expression which takes place in S cones a month before M/L cones. The majority of ribbon markers, presynaptic vesicular release and postsynaptic neurotransduction-related machinery is present in both plexiform layers by fetal week 13. By contrast, two crucial components for cone to bipolar cell glutamatergic transmission, the metabotropic glutamate receptor 6 and voltage-dependent calcium channel α1.4, are not detected until fetal week 22 when bipolar cell invagination is present in the cone pedicle. These results suggest an intrinsically programmed but nonsynchronous expression of molecules in cone synaptic development. Moreover, functional ribbon synapses and active neurotransmission at foveal cone pedicles are possibly present as early as mid-gestation in human retina.

Anolis carolinensis as a model to understand the molecular and cellular basis of foveal development.

The fovea is an anatomical specialization of the central retina containing closely packed cone-photoreceptors providing an area of high acuity vision in humans and primates. Despite its key role in the clarity of vision, little is known about the molecular and cellular basis of foveal development, due to the absence of a foveal structure in commonly used laboratory animal models. Of the amniotes the retina in birds of prey and some reptiles do exhibit a typical foveal structure, but they have not been studied in the context of foveal development due to lack of availability of embryonic tissue, lack of captive breeding programs, and limited genomic information. However, the genome for the diurnal bifoveate reptile species Anolis carolinensis (green anole) was recently published and it is possible to collect embryos from this species in captivity. Here, we tested the feasibility of using the anole as a model to study foveal development. Eyes were collected at various stages of development for histological analysis, immunofluorescence, and apoptosis. We show that at embryonic stage (ES) 10 there is peak ganglion cell density at the incipient central foveal region and a single row of cone photoreceptor nuclei. At ES17 the foveal pit begins to form and at this stage there are 3-4 rows of cone nuclei. Post-hatching a further increase in cone density and lengthening of inner and outer segments is observed. A yellowish pigment was seen in the adult central foveal region, but not in the temporal fovea. At ES14 Pax6 was localized across the entire retina, but was more prominent in the ganglion cell layer (GCL) and the part of the inner nuclear layer (INL) containing amacrine cell bodies. However, at ES17 Pax6 expression in the ganglion cells of the central retina was markedly reduced. Bioinformatic analysis revealed that 86% of human candidate foveal hypoplasia genes had an orthologous gene or DNA sequence in the green anole. These findings provide the first insight into foveal morphogenesis in the green anole and suggest that it could be a very useful model for investigating the molecular signals driving foveal development, and thus inform on human foveal development and disease.

The primate fovea: Structure, function and development.

A fovea is a pitted invagination in the inner retinal tissue (fovea interna) that overlies an area of photoreceptors specialized for high acuity vision (fovea externa). Although the shape of the vertebrate fovea varies considerably among the species, there are two basic types. The retina of many predatory fish, reptilians, and birds possess one (or two) convexiclivate fovea(s), while the retina of higher primates contains a concaviclivate fovea. By refraction of the incoming light, the convexiclivate fovea may function as image enlarger, focus indicator, and movement detector. By centrifugal displacement of the inner retinal layers, which increases the transparency of the central foveal tissue (the foveola), the primate fovea interna improves the quality of the image received by the central photoreceptors. In this review, we summarize ‒ with the focus on Müller cells of the human and macaque fovea ‒ data regarding the structure of the primate fovea, discuss various aspects of the optical function of the fovea, and propose a model of foveal development. The "Müller cell cone" of the foveola comprises specialized Müller cells which do not support neuronal activity but may serve optical and structural functions. In addition to the "Müller cell cone", structural stabilization of the foveal morphology may be provided by the 'z-shaped' Müller cells of the fovea walls, via exerting tractional forces onto Henle fibers. The spatial distribution of glial fibrillary acidic protein may suggest that the foveola and the Henle fiber layer are subjects to mechanical stress. During development, the foveal pit is proposed to be formed by a vertical contraction of the centralmost Müller cells. After widening of the foveal pit likely mediated by retracting astrocytes, Henle fibers are formed by horizontal contraction of Müller cell processes in the outer plexiform layer and the centripetal displacement of photoreceptors. A better understanding of the molecular, cellular, and mechanical factors involved in the developmental morphogenesis and the structural stabilization of the fovea may help to explain the (patho-) genesis of foveal hypoplasia and macular holes.

Molecular Anatomy of the Developing Human Retina.

Clinical and genetic heterogeneity associated with retinal diseases makes stem-cell-based therapies an attractive strategy for personalized medicine. However, we have limited understanding of the timing of key events in the developing human retina, and in particular the factors critical for generating the unique architecture of the fovea and surrounding macula. Here we define three key epochs in the transcriptome dynamics of human retina from fetal day (D) 52 to 136. Coincident histological analyses confirmed the cellular basis of transcriptional changes and highlighted the dramatic acceleration of development in the fovea compared with peripheral retina. Human and mouse retinal transcriptomes show remarkable similarity in developmental stages, although morphogenesis was greatly expanded in humans. Integration of DNA accessibility data allowed us to reconstruct transcriptional networks controlling photoreceptor differentiation. Our studies provide insights into human retinal development and serve as a resource for molecular staging of human stem-cell-derived retinal organoids.

A methodological approach for evaluation of foveal immaturity after extremely preterm birth.

PURPOSE: To characterize typical microanatomical alterations of immaturity in the fovea, that remain into childhood, after extremely preterm birth before 27 weeks gestational age (GA) and to suggest a clinical methodological evaluation tool. METHODS: Subjects were consecutively recruited at age 6.5 years and organized in four groups (10 subjects in each): Group A (full-term), Group B (GA 25-27 weeks) without retinopathy of prematurity (ROP), Group B* (GA 25-27 weeks) and Group C (GA 23-24 weeks) both with ROP stage 3. Foveal microanatomy was studied using a grading system of OCT-scans. RESULTS: Prematurely born children (including Group B, B* and C) had significantly reduced foveal depth (mean difference -53 µm, p < 0.001), thicker inner retinal layer (mean difference 21.6, p = 0.005) and thicker outer nuclear layer (mean difference 23.5, p < 0.001) than controls. Foveal depth and inner retinal layer thickness were significantly correlated to GA (p = 0.003 and p = 0.017 respectively) within the preterm group. Foveal depth increased with 14.1 µm per week between GA 23 and 27. The three hyper reflective bands of the outer segments as well as a central protuberance of inner and outer segment layers were present in all children. CONCLUSION: Previous studies have revealed signs of immaturity affecting most retinal layers at time of birth in prematurely born children. The present study adds information to which extent these signs of underdevelopment remains to later in life. The applied method showed that premature birth before GA 27 weeks commonly leads to characteristic anatomical alterations of the foveal anatomy expressed as reduced foveal depth and incomplete extrusion of the inner retinal layers. Although deviations of the outer nuclear layers can be seen in the most extremely preterm born children, the outer part of the fovea generally develops well, independent of prematurity. The single most important determinant for the degree of foveal maturation seems to be GA.

Evaluation of normal human foveal development using optical coherence tomography and histologic examination.

OBJECTIVE: To assess outer retinal layer maturation during late gestation and early postnatal life using optical coherence tomography and histologic examination. METHODS: Thirty-nine participants 30 weeks' postmenstrual age or older were imaged using a handheld optical coherence tomography system, for a total of 102 imaging sessions. Foveal images from 16 participants (21 imaging sessions) were normal and evaluated for inner retinal excavation and the presence of outer retinal reflective bands. Reflectivity profiles of central, parafoveal, and parafoveal retina were extracted and were compared with age-matched histologic sections. RESULTS: The foveal pit morphologic structure in infants was generally distinguishable from that in adults. Reflectivity profiles showed a single hyperreflective band at the fovea in all the infants younger than 42 weeks' postmenstrual age. Multiple bands were distinguishable in the outer retina at the peri fovea by 32 weeks' postmenstrual age and at the fovea by 3 months' postterm. By 17 months' postnatal, the characteristic appearance of 4 hyperreflective bands was evident across the foveal region. These features are consistent with previous results from histologic examinations. A "temporal divot" was present in some infants, and the foveal pit morphologic structure and the extent of inner retinal excavation were variable. CONCLUSIONS: Handheld optical coherence tomography is a viable technique for evaluating neonatal retinas. In premature infants who do not develop retinopathy of prematurity, the foveal region seems to follow a developmental time course similar to that associated with in utero maturation. CLINICAL RELEVANCE: As pediatric optical coherence tomography becomes more common, a better understanding of normal foveal and macular development is needed. Longitudinal imaging offers the opportunity to track postnatal foveal development among preterm infants in whom poor visual outcomes are anticipated or to follow up treatment outcomes in this population.

Histologic development of the human fovea from midgestation to maturity.

PURPOSE: To describe the histologic development of the human central retina from fetal week (Fwk) 22 to 13 years. DESIGN: Retrospective observational case series. METHODS: Retinal layers and neuronal substructures were delineated on foveal sections of fixed tissue stained in azure II-methylene blue and on frozen sections immunolabeled for cone, rod, or glial proteins. Postmortem tissue was from 11 eyes at Fwk 20-27; 8 eyes at Fwk 28-37; 6 eyes at postnatal 1 day to 6 weeks; 3 eyes at 9 to 15 months; and 5 eyes at 28 months to 13 years. RESULTS: At Fwk 20-22 the fovea could be identified by the presence of a single layer of cones in the outer nuclear layer. Immunolabeling detected synaptic proteins, cone and rod opsins, and Müller glial processes separating the photoreceptors. The foveal pit appeared at Fwk 25, involving progressive peripheral displacement of ganglion cell, inner plexiform, and inner nuclear layers. The pit became wider and shallower after birth, and appeared mature by 15 months. Between Fwk 25 and Fwk 38, all photoreceptors developed more distinct inner and outer segments, but these were longer on peripheral than foveal cones. After birth the foveal outer nuclear layer became much thicker as cone packing occurred. Cone packing and neuronal migration during pit formation combined to form long central photoreceptor axons, which changed the outer plexiform layer from a thin sheet of synaptic pedicles into the thickest layer in the central retina by 15 months. Foveal inner and outer segment length matched peripheral cones by 15 months and was 4 times longer by 13 years. CONCLUSIONS: These data are necessary to understand the marked changes in human retina from late gestation to early adulthood. They provide qualitative and quantitative morphologic information required to interpret the changes in hyper- and hyporeflexive bands in pediatric spectral-domain optical coherence tomography images at the same ages.

Foveal shape and structure in a normal population.

PURPOSE: The shape of the human fovea presents important but still poorly characterized variations. In this study, the variability of the shape and structure of normal foveae were examined. METHODS: In a group of 110 eyes of 57 healthy adults, the shape and structure of the fovea were analyzed by automated segmentation of retinal layer on high-resolution optical coherence tomography scans. In an additional group of 10 normal eyes of 10 patients undergoing fluorescein angiography, the size of the foveal avascular zone (FAZ) was correlated to foveal shape. RESULTS: From the thickest to the thinnest fovea, there was a structural continuum ranging from a shallow pit with continuity of the inner nuclear layer (INL) over the center (seven eyes; 6.7%), to a complete separation of inner layers overlying a flat and thinner central outer nuclear layer (ONL; eight eyes; 7.3%). Central foveal thickness correlated inversely to the degree of inner layer separation and to the surface of the FAZ. CONCLUSIONS: Foveal structure strongly correlates with its neurovascular organization. The findings support a developmental model in which the size of the FAZ determines the extent of centrifugal migration of inner retinal layers, which counteracts in some way the centripetal packing of cone photoreceptors.

The foveal avascular region of developing human retina.

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.

The developing and evolving retina: using time to organize form.

Evolutionary and other functional accounts of the retina and its normal development highlight different aspects of control of its growth and form than genomic and mechanistic accounts. Discussing examples from opsin expression, developmental regulation of the eye's size and optical quality, regulation of eye size with respect to brain and body size, and the development of the fovea, these different aspects of control are contrasted. Contributions of mouse models, particularly with regard to relative timing of events in different species are reviewed, introducing a Web-based utility for exploration of timing issues (www.translatingtime.net). Variation at the individual level, in early experience, and also across species is an essential source of information to understand normal development and its pathologies.

Development of the human retina in the absence of ganglion cells.

Retinal development was studied in eyes from fetal and neonatal human anencephalic (AnC) and normal age-matched infants to determine the time of retinal ganglion cell (GC) loss and its effect on the development of other retinal neurons. At fetal week (Fwk) 14, GC loss was evident in central retina and by Fwk 19-20 almost all GC were absent, although immunocytochemical labeling for GC markers brain 3, neurofilament M and parvalbumin detected a few GC in the AnC far periphery at older ages. The inner nuclear and inner plexiform (IPL) layers showed variable amounts of thinning but all normal bipolar (BP) and horizontal cell markers were still present. The amacrine (AM) labels calbindin and calretinin were markedly reduced. Lamination for these markers in the IPL was less organized than in normal retinas, with BP and AM markers extending into the degenerated GC layer. Cone and rod photoreceptors had normal morphology and topography in AnC retina and each expressed normal phototransduction and synaptic markers. The prospective fovea was identified in AnC neonatal retina by cone packing and the absence of immunolabeled rod photoreceptors. In one AnC neonatal retina, blood vessels and astrocytes extended across the inner retina in the putative fovea and there was no evidence of a pit. In another AnC neonatal retina, blood vessels and astrocytes formed a foveal avascular zone in the inner retina and a shallow pit was present within this zone. However, both foveas showed evidence for the onset of cone elongation and packing. These findings support the model of Springer and Hendrickson [2005; Vis. Neurosci. 22, 171] in which the foveal avascular zone is critical for pit formation, but suggest that mechanisms inherent to the outer retina may be involved in early stages of foveal cone packing.

Development of the primate area of high acuity, 3: temporal relationships between pit formation, retinal elongation and cone packing.

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.

The Pax6 isoform bearing an alternative spliced exon promotes the development of the neural retinal structure.

The vertebrate retina has an area where visual cells are closely packed for proper vision that is known as a fovea, an area centralis or a visual streak. The molecular mechanism that regulates the formation of these structures and visual cell gradients is unknown. The transcription factor Pax6 is a master regulator of eye development. A Pax6 isoform that contains an exon 5a-encoded 14 amino acid insertion in its paired domain, Pax6(+5a), has different DNA-binding properties compared with the Pax6(-5a) isoform. Little is known about the functional significance of Pax6(+5a). Here, we show that Pax6(+5a) is expressed especially in the retinal portion where visual cells accumulate during eye development and, when overexpressed, induces a remarkable well-differentiated retina-like structure. Pax6(+5a) proteins that bear point mutations that are found in patients with foveal hypoplasia are unable to induce these ectopic retina-like structures. We propose that Pax6(+5a) induces a developmental cascade in the prospective fovea, area centralis or visual streak region that leads to the formation of a retinal architecture bearing densely packed visual cells.

Differential distribution of fibroblast growth factor receptors (FGFRs) on foveal cones: FGFR-4 is an early marker of cone photoreceptors.

PURPOSE: Relatively little is known of the expression and distribution of FGF receptors (FGFR) in the primate retina. We investigated expression of FGFRs in developing and adult Macaca monkey retina, paying particular attention to the cone rich, macular region. METHODS: One fetal human retina was used for diagnostic PCR using primers designed for FGFR1, FGFR2, FGFR3, FGFR4, and FGFR like-protein 1 (FGFrl1) and for probe design to FGFR3, FGFR4, and FGFrl1. Rat cDNA was used to synthesize probes for FGFR1 and FGFR2 with 90% and 93% homology to human, respectively. Paraffin sections of retina from macaque fetuses sacrificed at fetal days (Fd) 64, 73, 85, 105, 115, 120, and 165, and postnatal ages 2.5 and 11 years were used to detect FGF receptors by immunohistochemistry and in situ hybridization. RESULTS: PCR showed each of the FGF receptors are expressed in fetal human retina. In situ hybridization indicated that mRNA for each receptor is expressed in all retinal cell layers during development, but most intensely in the ganglion cell layer (GCL). FGFR2 mRNA is reduced in the adult inner (INL) and outer (ONL) nuclear layers, while FGFrl1 mRNA is virtually absent from the adult ONL. FGFR4 mRNA is particularly intense in fetal and adult cone photoreceptors. Immunoreactivity to FGFR1-FGFR4 was detected in the interphotoreceptor matrix in what appeared to be RPE microvilli associated with developing photoreceptor outer segments, and generally is high in the GCL and low in the INL. Different patterns of FGFR3 and FGFR4 immunoreactivities in the outer plexiform layer (OPL) suggest localization of FGFR3 to horizontal cell processes, with FGFR4 being expressed by both horizontal and bipolar cell processes. FGFR1, FGFR3, and FGFR4 immunoreactivities are present in the inner segments and somata of adult cones. The pedicles of developing and adult cones are FGFR1 and FGFR3 immunoreactive, and the basal, synaptic region is FGFR4 immunoreactive. FGFR4 labels cones almost in their entirety from early in development and is not detected in rods. The fibers of Henle are intensely FGFR4 immunoreactive in adult cones. CONCLUSIONS: The results show high levels of FGF receptor expression in developing and adult retina. Differential distribution of FGF receptors across developing and adult photoreceptors suggests specific roles for FGF signalling in development and maintenance of photoreceptors, particularly the specialized cones of the fovea.

VEGF expression by ganglion cells in central retina before formation of the foveal depression in monkey retina: evidence of developmental hypoxia.

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.

Developmental changes in astrocyte density in the macaque perifoveal region.

We studied astrocyte density both in the perifoveal region and in extrafoveal regions within the same distance of the optic disc (OD) over a time period from foveal pit formation (embryonic day E112) until 2 months after birth. The study was prompted by earlier observations that the adult macaque displays an almost astrocyte-free region around the fovea which, however, at birth is occupied by astrocytes. Thus, we wanted to determine if the perifoveal region is invaded by astrocytes during early development to the same degree as other regions in the central retina, and how the reduction in density can be explained. From the earliest age we studied (embryonic day 112), less astrocytes were found in the perifovea than in other regions equidistant from the OD. In addition, the number of astrocytes steadily declined both in the perifovea and outside until birth. During the first week after birth, there was a further dramatic decline in perifoveal astrocyte density. Double-labelling with glial fibrillary acidic protein (GFAP) immunocytochemistry and the TUNEL method showed that during the whole observation period astrocytes undergo DNA fragmentation and presumably die. However, the rate of TUNEL-positive astrocytes did not significantly differ between perifovea and other regions equidistant to the OD, and at no time did we find a significant peak of apoptosis rate. Thus, the reduction in perifoveal astrocyte density cannot be explained by missing invasion or by selectively elevated apoptosis rates in the foveal and perifoveal regions. Alternative hypotheses are discussed.

Apoptosis during development of the human retina: relationship to foveal development and retinal synaptogenesis.

Apoptosis in the ganglion cell (GCL) and inner nuclear (INL) layers of human fetal retinae aged 14-35 weeks of gestation (WG) was investigated in relation to synaptogenesis and foveal depression formation. Terminal transferase dUTP-biotin nick end labeling (TUNEL) was used to identify apoptosis, and synapse development was demonstrated by synaptophysin immunoreactivity (-IR). The distribution of apoptotic cells and synaptophysin-IR was studied as a function of eccentricity. Between 14 and 23-24 WG in the GCL, rates of apoptosis were relatively low in central retina. A shallow fovea was detected at 23-24 WG. In the central GCL, the rate of apoptosis was 0.21% of viable cells compared with a higher incidence of 0.79-1.64% peripherally. Apoptosis in the INL was 2-8 times greater than that in the GCL. At 14-15 WG, peak death occurred at the incipient fovea; however, by 20 WG the distribution was bimodal, with peaks at more eccentric locations on either side of the incipient fovea with increasing age. Approximately 90% of INL apoptotic cells were in the middle and outer regions, suggesting that bipolar cells formed the majority of dying neurons. Synaptophysin-IR was present in cones, bipolar cells, and processes in the inner and outer plexiform layers at the incipient fovea at 14 WG and spread peripherally with increasing age. The peripheral margin of synaptophysin-IR coincided with areas of peak INL apoptosis. This pattern suggests that bipolar cell elimination is associated with the onset of synaptogenesis. Apoptosis in the GCL and INL is not a significant factor in foveal depression morphogenesis.

Maturational gradients in the retina of the ferret.

In the present set of studies, we have examined the site for the initiation of retinal maturation in the ferret. A variety of maturational features across the developing inner and outer retina were examined by using standard immunohistochemical, carbocyanine dye labelling, and Nissl-staining techniques, including 1) two indices of early differentiation of the first-born retinal ganglion cells, the presence of beta-tubulin and of neuron-specific enolase; 2) the receding distribution of chondroitin sulfate proteoglycans within the inner retina; 3) the distribution of the first ganglion cells to grow axons along the optic nerve; 4) the emergence of the inner plexiform layer; 5) the emergence of the outer plexiform layer and 6) the onset of synaptophysin immunoreactivity within it; 7) the differentiation of calbindin-immunoreactive horizontal cells; and 8) the cessation of proliferative activity at the ventricular surface. Although we were able to define distinct maturational gradients that are associated with many of these features of inner and outer retinal development (each considered in detail in this report), with dorsal retina maturing before ventral retina, and with peripheral retina maturing last, none showed a clear initiation in the region of the developing area centralis. Rather, maturation began in the peripapillary retina dorsal to the optic nerve head, which is consistent with previous studies on the topography of ganglion cell genesis in the ferret. These results make clear that the order of retinal maturation and the formation of the area centralis are not linked, at least not in the ferret.