Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

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Glossary

Birthdate or born – The time when a progenitor permanently exits the cell cycle. Marking cells in their final division is referred to as birthdating.

Cell fate determination – The process by which a progenitor is programmed to become a specific cell type and its functional maturation. In the retina, a progenitor needs to acquire competence, exit the cell cycle, become specified, and differentiate. Competence – The potential to adopt a certain cell fate(s).

Differentiation – The functional maturation of a specified cell.

Histogenesis – The development of a mature tissue from a naive progenitor population.

Lineage – A cohort of cells derived from division(s) of a common progenitor. This is also referred to as a clone. Lineage tracing – The use of an indelible marker to label cells and all their descendants. Analysis at a later time point allows the inference of lineal relationships. This is also referred to as fate mapping.

Multipotent progenitor/progenitor – A cell that has competence to adopt several different fates. These cells may or may not be proliferative.

Progressive restriction model – A model of cell fate determination where multipotent progenitors lose the competence to adopt multiple cell fates over time. By this model, early progenitors have the competence to form all fates in a tissue.

Serial competence model – A variation on the progressive restriction model whereby multipotent progenitors transiently gain and subsequently lose competence for a subset of fates in a tissue over time. By this model, progenitors do not have the competence to form late fates at the earliest time points.

Specification or commitment – The point when a progenitor cell has irreversibly decided on a cell fate.

Birthdating

An interesting property of retinal cells is that they do not continue to divide after they differentiate. This means

that at some point in development, a progenitor permanently exits the cell cycle, referred to as its birthdate. Investigators took advantage of this property and designed a clever pulse-chase experiment to investigate whether different cell types exited the cell cycle at characteristic times. Animals at various stages of development were given a pulse of 3[H]-thymidine (3HdT). The 3HdT is incorporated into replicating DNA during synthesis

(S)-phase and any excess is cleared from the body quickly. The incorporated 3HdT is maintained in the newly synthesized DNA permanently. If the cell continues to divide, it will dilute the 3HdT signal by one-half each division. Next, autoradiography was conducted after the retina was fully formed (the chase). Cells that retained maximum labeling are those that exited the cell cycle (born) on the day of 3HdT administration. More recently, birthdating studies have been conducted with synthetic nucleotides, such as 5-bromo-2-deoxyuridine (BrdU), which can be detected by antibodies instead of autoradiography.

About 50 years ago, Sidman used birthdating studies to test what order, if any, retinal cells were formed in the rodent retina. The observations of Sidman and future investigators revealed a stereotypical birth order that was broadly broken down into early and late groups (Figure 1). Retinal ganglion cells (RGCs) were born first, followed closely by horizontal cells, cones, and amacrines. The late cohort comprised rods, bipolar cells, and Mu¨ller glia. Birthdating has been conducted in several vertebrate species. While there are some small differences in the order, the first cells born in all species examined are RGCs. Although there is clearly an overall birth order, which is evident from the production onset of each cell type, there is also considerable overlap in the genesis of the cell types, such that multiple cell types are born on the same day of development (Figure 1). This overlap is also observed in species where retinal histogenesis is long, such as in monkey. These data implied that cell fate determination is not strictly regulated by measuring time (or cell cycles) during development. Nonetheless, the observance of a birth order indicated that there is a temporal input into cell fate determination.

These studies raised several questions about retinal progenitors. Are all retinal cell types derived from the progenitors in the optic cup? Are there different progenitors for each retinal cell type? Is fate choice predetermined or stochastic? These questions were addressed by tracing the fate of individual progenitors.

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Lineage Tracing

To understand the behavior of progenitor cells, a way to trace the fate of individual progenitors was needed. Starting in the late 1980s, investigators designed elegant lineagetracing (fate-mapping) experiments to study the retina. In rodents, Turner, Snyder, and Cepko used replicationincompetent retroviruses encoding a marker gene (e.g., LacZ) to infect retinal progenitors at various time points. These retroviruses can only infect dividing progenitors. Since the virus integrates into the genome, the progenitor and its descendents become permanently labeled. By adjusting the titer of the virus, individual progenitor lineages (clones) can be mapped from mature retinas (Figure 2).

The first set of retroviral lineage-tracing studies examined postnatal infections of rat retinas. In postnatal day 0 (P0) infections, majority of clones contained rods and were predominantly small (one to four cells). A large number of clones contained only rods, whereas others contained rods, amacrines, bipolars, and/or Mu¨ller glia (Figure 2 and Table 1). There were few clones that did not contain any rods. Cell types born before P0 (e.g., cones, horizontals, and RGCs) were not observed in these clones, consistent with previous birthdating analyses. Clones generated from P2, P4, and P7 infections become progressively smaller. This reflects the progressive decrease in progenitor division that occurs in the first postnatal week. Clones were heterogeneous; some

contained multiple cell types and others one cell type or just a single cell. From all these time points, there was no obvious lineage hierarchy that could be constructed from the clone composition data. This showed that mammalian retinal cell fate determination is stochastic, or nondeterministic. Importantly, they observed two-cell clones that had different fates. This implied that fate choice is decided during or after the last cell division.

To see if the progenitors in the optic cup can give rise to all the retinal cell types, the investigators generated clones from embryonic (E) time points when few cells have exited the cell cycle. For this reason, they injected their retroviruses into the subretinal space of E13 and E14

mice in utero, a difficult procedure. The mice were allowed to mature and clone composition was examined. The clone size and composition were highly heterogeneous (Table 1). All seven cell fates were represented in these clones. Moreover, these clones never contained any other cell types (i.e., astrocytes, vascular endothelial cells, pigment epithelium, etc.). These early lineage traces showed that all seven retinal cell types derive solely from a retina-restricted progenitor pool. The heterogeneity in clone composition (there were few multicell clones alike) reinforced that fate choice is apparently stochastic. Consistent with retinal cell frequency ( 78% are rods in mice), nearly all clones contained one or more rods. Clone size varied from 1 to

234 cells and had a bimodal distribution. There were abundant small clones (<5 cells) and abundant large clones (>20 cells). The largest clones are simply too big to have been generated by simple asymmetric (one progenitor, one neuron) divisions in the time allotted for development. Based on the distribution of clone sizes, it is possible that progenitors preferentially utilize symmetric divisions during retinal histogenesis. Lineage-tracing studies have been conducted in other vertebrates, yielding similar results.

These lineage-tracing studies answered several questions. First, retinal progenitors in the optic cup are multipotent and give rise to all seven cell types. Second, there are no separate progenitors for each cell type. Nonetheless, rod-only clones were observed, raising the possibility of a rod-only progenitor. Since rods make up 78% of the mouse retina, smallto medium-sized rod-only clones are expected from multipotent progenitors. Fourth, cell fate choice is stochastic. Fifth, cell fate specification occurs during the last cell cycle or later. These data raised several more questions about the mechanisms of retinal histogenesis. Is fate determination a cell autonomous process, a cell nonautonomous process, or a combination of both? Do retinal progenitors have broad competence that is gradually lost, or are progenitors more limited in their cell fate choices during development? These questions have been addressed by manipulating the local cellular environment.

Environmental Challenge

Several approaches have been undertaken to discriminate between cell autonomous and cell nonautonomous control of retinal cell fate determination. Most of these experiments involve challenging retinal progenitors with different environments. Unlike the previous birthdating and lineage-tracing studies, these environmental challenge experiments are harder to interpret and, at times, yield conflicting results.

One type of experiment was designed to answer the question: What is the default state of retinal progenitors? For these experiments, retinal progenitors were labeled with 3HdT and dissociated to single cell density to examine their potential for differentiation in isolation. Reh and Kljavin saw that when rat progenitors were isolated from early stages of development, the majority of the progenitors differentiated into RGCs, while progenitors isolated from late stages of development differentiated into cell types normally generated late in development, like rods. A somewhat different result was obtained from similar studies in the chick embryo by Adler and colleagues, suggesting that cones were the default cell fate. However, subsequent studies in chick revealed a ganglion cell bias for the progenitors isolated from the earliest stages of

retinal development. Together, these results led to the concept of a rolling default, or shifting competence in the progenitors; in other words, there is an intrinsic bias to the types of neurons generated by the progenitors that shifts progressively over developmental time. This idea has been recently supported by Notch-signaling studies. The Notch receptor is active in progenitor cells throughout development, and inhibition of signaling leads to premature progenitor differentiation. Inhibition of Notch in early progenitors leads to the overproduction of RGCs and cones, whereas inhibition of Notch function later in development results in overproduction of rods, but not of RGCs or cones. These results show that retinal progenitors change their competence intrinsically over time.

In the next type of experiment, investigators asked whether inductive interactions among the retinal cells played a role in their cell fate determination. Studies from Drosophila eye imaginal disk had shown an important role for cell–cell interactions in directing the ommatidial progenitors to their individual identities. In that tissue, the data were best fit by a sequential cell induction model in which the first cells, the R8 photoreceptors, induce the recruitment of the next type of photoreceptor, and so on. To test whether similar sequential inductions occurred in vertebrate retinas, investigators used heterochronic co-cultures, surrounding early embryonic progenitors with late-generated retinal cells. The groups of Raff and Reh co-cultured early embryonic rodent retinal cells with an excess of postnatal retinal cells. In both sets of experiments, the early progenitors were more likely to develop into rhodopsin expressing (rod photoreceptors) cells when compared to early progenitors cultured alone (which developed primarily early retinal fates). This increase was not seen when the challenging (older) cells were derived from the brain instead of the retina. Watanabe and Raff also found that the increase in rods was observed when the two cell populations were separated by a cell-impermeable membrane. Together, these data showed that a soluble factor(s) from late retina can promote rod fate in younger cells. In addition, very early mouse retinal progenitors (E11–E12) could form rods when cultured with an excess of older rat retinal cells; birthdating analyses indicate that rods are not normally generated by progenitors from this very early retina, which suggests that rod competence precedes rod genesis by at least 1 day. These experiments led to the idea that although progenitors may have an intrinsic bias in the types of cells they generate at any time in development, their fate can be influenced by factors in the microenvironment. Many subsequent studies have identified signaling factors in the developing retina that can influence the fate of the progenitors and, particularly, factors that can increase the percentage of these cells that differentiate into rods; however, since low-density cultures do not support robust rod differentiation (i.e., expression of

rhodopsin and other markers), it is possible that most of the factors identified to date have more of an effect on the expression of these identifiers, rather than the choice of the rod fate per se.

Another concept that emerged from the early studies of retinal development was the idea that specific cell types use feedback regulation to control their density. Elimination of dopaminergic amacrine cells in the developing frog leads to an overproduction of these cells by progenitors at the ciliary marginal zone. This suggested that there is a nonautonomous feedback regulation to negatively control amacrine cell number. In vitro experiments found a similar effect in developing rat retina. When E16 retinal cells were co-cultured with an excess of P0 cells, fewer amacrines were generated. However, when the P0 cells were depleted of amacrine cells, there was an increase in the number of amacrine cells generated by the E16 cells. When the converse experiment was done, P0 cells co-cultured with an excess of E16 cells, an increase in amacrines and bipolars was seen along with a decrease in rods. This confirmed the presence of negative feedback on amacrine cells and implied feedback regulation of bipolar cell genesis. The concept of feedback regulation of retinal cell production was extended to ganglion cells by Waid and McLoon. They examined the influence of a late retinal environment on RGC fate determination using heterochronic co-cultures in chick. Early cells were inhibited from RGC fate when co-cultured with late retinal cells. This inhibition was not observed when RGCs were depleted from the challenging (late) cell population, which showed that RGCs can nonautonomously feed back to inhibit further RGC production. In a subsequent study from the McLoon lab, they blocked Notch signaling (an RGC inhibitor) at different times. Interestingly, when later time points were examined, they saw newborn RGCs in areas where RGC genesis had ceased in controls. This suggested that RGC competence extends beyond RGC genesis.

Although the studies described above have supported a role for cell–cell interactions in the regulation of cell fate in the vertebrate retina, some types of studies have failed to find nonautonomous effects. Rapaport and colleagues conducted heterochronic transplants in frogs. When younger retinal tissue was transplanted into older hosts, the donor tissue did not adopt later fates or differentiate early. This result was also seen in co-cultured cells in vitro. Importantly, the donor cells directly adjacent to the older host cells were not fate shifted. This suggested that cell fate determination is not regulated by changing environmental stimuli, rather, that cell competence is limiting in frogs. Cayouette and colleagues combined lineage tracing and single cell culturing of rodent progenitor cells. In this experiment, E16–E17 rat progenitors were plated at single cell density and the resulting clones were examined 7–10 days later. In parallel, they conducted a retroviral

lineage trace (similar to above) in E16–E17 retinal explants (intact tissue) cultured the same amount of time. The clones in both cases were screened for rod, bipolar, amacrine, and Mu¨ller glial cell fates. The clone composition and size in both experimental systems were similar in isolation and in explants. This suggested that fate choice is largely cell autonomous and that any given cell type is not required to induce (specify) another. In sum, while many studies have shown a role for cell–cell interactions in the control of retinal cell fate, the relative importance of intrinsic and extrinsic regulation is still not resolved.

These data have been used to assemble cell fate determination models (Figure 3). One model, progressive restriction, argues that early progenitors have competence to adopt all retinal cell fates and that this competence is gradually lost (restricted) over time. By this model, nonautonomous contributions are expected to specify cell fate in these multipotent progenitors over time. Early progenitors should be able to adopt late fates (if stimulated properly) and competence should extend beyond the normal genesis window to allow for feedback inhibition. Another model, serial competence, contends that progenitors have competence for only a few cell fates at any given time and that progenitors serially cycle through numerous restricted competence states. By this model, cell nonautonomous inputs are not required for fate specification. Presumably, the mechanism that controls competence dynamics would be reflected by the complement of transcription factors expressed in progenitors. In the next section, we discuss evidence that transcription factors regulate competence.

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Figure 3 Cell fate determination models. (a) Serial competence model. The progenitor changes over time in their potential to generate different types of retinal cells. These changes in competence would be mediated by changes in the complement of transcription factors present in the cells. (b) Progressive restriction model. The progenitor can initially generate all types of retinal neurons, but over time loses one or another of the transcription factors needed for a specific fate. This is shown in the figure as what is initially a rainbow-colored cell, progressing into a cell that is only red. In principle, the progressive restriction could be due to a loss in key transcription factors, or alternatively by the addition of new repressors.

Transcription Factors and Competence

How transcription factors regulate retinal progenitor competence is beginning to be characterized (Figure 4). Although most of these transcription factors are expressed in the postmitotic cells, and therefore are unlikely to regulate the competence of the progenitor cells per se, there are members of several different transcription factor families that are expressed in the mitotically active cells. One transcription factor expressed by progenitors that appears to convey competence is Pax6. This eye-field transcription factor is expressed in all progenitors and directly promotes the expression of other transcription factors (Ascl1, Ngn2, Atoh7, etc.). Specific deletion of Pax6 from progenitors leads to the apparent loss of competence to generate all retinal cell types except amacrine cells. A complementary result is obtained when FoxN4, a transcription factor expressed in a subpopulation of progenitors, is deleted in mice. In FoxN4 null mice, amacrine cells fail to develop. This suggests that the combination of FoxN4 and Pax6 can convey progenitors with competence for all retinal cell fates. Ikaros is a transcription factor that is expressed primarily by early-staged progenitors, and mice deficient in this gene have fewer early-born neurons.

While these examples show that changes in transcription factors can affect the types of neurons produced by

Figure 4 Transcription factor code. Kageyama and others have proposed that a combination of bHLH and homeodomain transcription factors specify each retinal cell type. Although most of these factors are expressed primarily in postmitotic cells, and therefore might not be candidates for the changing competence models described above, several lines of evidence indicate that these factors are necessary for the full differentiation of these cell types.

progenitors, and hence their competence to generate specific neuronal types in the retina, it has also become clear that a more general level of competence, to generate neurons versus glia, is also conveyed by these factors. Another transcription factor expressed in a subset of progenitors is Ascl1 (Mash1), a member of the bHLH class. Prior to E15 in the rat retina, there is little Ascl1 expression in the retina, though progenitors by this age are producing RGCs, cones, rods, and horizontal and amacrine cells. Even after E15, Ascl1 is expressed in most, but not all, progenitors. Deletion of this gene in mice leads to an overproduction of Mu¨ller glia, apparently at the expense of rods and bipolar cells. It appears that Ascl1 imparts retinal progenitors with the competence for lategenerated retinal neurons, while also inhibiting Mu¨ller glial fate specification (Figure 5) in part by maintaining the expression of Hes6, but also by driving expression of key components of the Notch pathway, including Hes5 and Hes1, to maintain the progenitors in an undifferentiated state while they generate additional neurons. When Notch signaling is reduced, even for times as short as 6 hours, the retinal progenitors are irreversibly committed to exit the cell cycle and differentiate. Since the Notch effector genes, Hes1 and Hes5 are normally regulated during the cell cycle, such they are lowest during the G2 and M-phases, one model for the mechanism by which progenitors initiate differentiation is through a progressive slowing of the cell cycle as development proceeds (Figure 5). Immunolabeling for the active Notch intracellular domain in mouse retina has confirmed that Notch signaling is lowest in cells whose nuclei are at the apical surface. However, this is apparently not the case in the fish retina. Live imaging studies in cortex by Kageyama’s group confirm that Notch signaling oscillates through the cell cycle, with the lowest levels in the progenitors with nuclei located at the apical surface, those cells that are in G2or M-phases of the cell cycle. Given the highly transient nature of Notch signaling and the very fast cell cycles in fish, further studies using highly destabilized reporters will be needed in the fish to determine whether this difference is real.

Another member of the bHLH class of transcription factors, Atoh7 (Math5), is transiently expressed by a small subset of postmitotic progenitors. Lineage-tracing and genetic-deletion studies have shown that it is necessary for RGC competence. In addition to these transcription factors expressed in progenitors, there are many that are expressed in subpopulations of nascent neurons. For example, Ptf1a, NeuroD1, and Math3 are expressed in amacrine cells, and loss of one or more of these genes leads to defects in amacrine cell fate determination or survival. Moreover, overexpression of transcription factors such as NeuroD1 can drive progenitor differentiation into specific cell types, though the types appear to vary depending on the species and the method of

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overexpression. These studies show that few transcription factors fit cleanly in the simple models of progressive restriction or serial competence. Moreover, since few targets of these transcription factors have been identified, the nature of competence regulation remains unclear. Nevertheless, the transcription factor networks for two cell types, RGCs and rod photoreceptors, are beginning to be worked out.

Conclusions

A great deal has been done over the past 50 years to understand the developmental mechanisms of vertebrate retinal histogenesis. Birthdating studies have revealed a characteristic genesis order of the retinal cell types. Lineage-tracing experiments have shown that retinal progenitors are multipotent and that fate choice is stochastic.

Experiments that challenge the environment of retinal progenitors have revealed that both cell autonomous and cell nonautonomous factors contribute to fate determination. More recent techniques, such as expression fate mapping (lineage by gene expression) and live imaging of cell lineages, will further our understanding of retinal fate determination. In addition, the recent increase in early cell-type-specific markers will allow us to more precisely examine the effects of environmental challenges. While several factors have been identified, many more experiments are needed to elucidate the molecular mechanisms of retinal fate determination.

See also: Coordinating Division and Differentiation in Retinal Development; Ganglion Cell Development: Early Steps/Fate.

Further Reading

Cayouette, M., Barres, B. A., and Raff, M. (2003). Importance of intrinsic mechanisms in cell fate decisions in the developing rat retina. Neuron 40(5): 897–904.

Elliott, J., Jolicoeur, C., Ramamurthy, V., and Cayouette, M. (2008). Ikaros confers early temporal competence to mouse retinal progenitor cells. Neuron 60(1): 26–39.

Li, S., Mo, Z., Yang, X., et al. (2004). Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors. Neuron 43(6): 795–807.

Marquardt, T., Ashery-Padan, R., Andrejewski, N., et al. (2001). Pax6 is required for the multipotent state of retinal progenitor cells. Cell 105(1): 43–55.

Nelson, B. R., Hartman, B. H., Ray, C. A., Hayashi, T., BerminghamMcDonogh, O., and Reh, T. A. (2009). Acheate-scute like 1 (Ascl1) is required for normal delta-like (Dll) gene expression and notch signaling during retinal development. Development Dynamics 238: 2163–2178.

Ohsawa, R. and Kageyama, R. (2008). Regulation of retinal cell fate specification by multiple transcription factors. Brain Research 1192: 90–98.

Rapaport, D. H., Wong, L. L., Wood, E. D., Yasumura, D., and LaVail, M. M. (2004). Timing and topography of cell genesis in the rat retina.

Journal of Comparative Neurology 474: 304–324.

Reh, T. A. (1992). Cellular interactions determine neuronal phenotypes in rodent retinal cultures. Journal of Neurobiology 23: 1067–1083.

Reh, T. A. and Kljavin, I. J. (1989). Age of differentiation determines rat retinal germinal cell phenotype: Induction of differentiation by dissociation. Journal of Neuroscience 9(12): 4179–4189.

Shimojo, H., Ohtsuka, T., and Kageyama, R. (2008). Oscillations in notch signaling regulate maintenance of neural progenitors. Neuron 58: 52–64.

Turner, D. L. and Cepko, C. L. (1987). A common progenitor for neurons and glia persists in rat retina late in development. Nature 328: 131–136.

Turner, D. L., Snyder, E. Y., and Cepko, C. L. (1990). Lineageindependent determination of cell type in the embryonic mouse retina. Neuron 4: 833–845.

Waid, D. K. and McLoon, S. C. (1998). Ganglion cells influence the fate of dividing retinal cells in culture. Development 125: 1059–1066.

Watanabe, T. and Raff, M. C. (1992). Diffusible rod-promoting signals in the developing rat retina. Development 114: 899–906.

Glossary

Angiogenesis – The formation of new blood vessels from preexisting ones.

Atrophy – To wither or deteriorate.

Blood–retinal barrier – Specialized, nonfenestrated, tightly joined endothelial cells that form a transport barrier for certain substances between the retinal capillaries and the retinal tissue.

Choroidal neovascularization – A condition whereby new blood vessels that originate from the choroid grow and break through Bruch’s membrane into the subretinal pigment epithelium (sub-RPE) or subretinal space.

Dominant negative – A genetic mutation where the gene product adversely affects the normal, wild-type gene product within the same cell.

Fenestration – A small pore (60–80 nm in diameter) within the endothelium wall that allows the passage of small molecules and a limited amount of proteins. Microphthalmia – An abnormal smallness of the eyes, occurring as the result of disease or of imperfect development.

Phagocytosis – The process in which phagocytes engulf and digest microorganisms and cellular debris.

Quiescence – The absence of proliferation. Trophic factor – A molecule that promotes cellular growth and/or survival.

Introduction

Located at the back of the eye, the retinal pigment epithelium (RPE)–choroid complex is comprised of the RPE, a polarized, epithelial monolayer and the choroid, a highly fenestrated vascular bed. Separated by Bruch’s membrane (BrM), an elastic lamina, the RPE and choroid each play a vital role in normal eye physiology. Considerable evidence indicates that there is not only a great deal of interaction between the RPE and choroid, but that the integrity of this interaction is critical to normal eye function. Examination of the morphology of the choroidal microvasculature reveals that the vessels are ‘‘polarized,’’ with fenestrations preferentially localized to the capillary surface proximal to the RPE and BrM. Whereas the

cytoplasm of the endothelial cell is thinnest in this region, endothelial cell bodies and nuclei are more prominent distal to the RPE. These observations led to the speculation that the RPE exerts an inductive effect on the choriocapillaris by releasing a factor that diffuses across BrM to provide a trophic effect and to mediate the anatomic specializations in the capillary endothelial cells. Preservation of a normal interaction between the RPE and choroid is required for proper eye physiology. This review explores the intricacies of the RPE and choroid, and their interactions during development, in the adult and with aging.

RPE–Choroid Complex Development

RPE Development

The development of the RPE is dependent upon the coordination of transcription factor expression and inductive signals received from the tissues surrounding the developing eye. Eye development proceeds from two principal tissue components: the neural ectoderm, which buds from the wall of the forebrain to form the optic vesicle, and the surface ectoderm, which forms the lens (Figure 1). When the optic vesicle comes in contact with the surface ectoderm, it invaginates, forming the optic cup. The optic cup consists of an inner layer, which gives rise to the neural retina, and an outer layer, which forms the RPE. At the optic cup stage, the presumptive RPE and retina are separated by a thin remnant of lumen, which becomes filled by a material known as the interphotoreceptor matrix (IPM). Coincident with this is the onset of the expression of RPE65, which encodes a protein involved in the conversion of all-trans-retinal to 11-cis- retinal. Prior to this stage, the RPE is a ciliated and pseudo-stratified epithelium; however, following IPM formation, RPE maturation commences. The onset of RPE maturation is marked by melanogenesis, which requires the activation of the tyrosinase promoter.

As the RPE continues to differentiate, it displays a complete apical to basolateral polarity, with short apical microvilli and small basolateral membrane infoldings, and the formation of tight junctions between the RPE cells, which can be divided into three stages. The early stage of tight junction formation is characterized by the expression of key tight junction proteins, such as zona occludens 1 (ZO-1), occludin, and claudins. However, these tight junctional complexes are rudimentary so the RPE lacks complete barrier properties and is therefore leaky. As the