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

(b)Retinal illuminance (log photopic trolands)

Figure 6 Mean root mean square (rms) wavefront aberration (a parameter that relates to the quality of the retinal image) was measured in 26 subjects and is plotted as a function of pupil diameter. The error bars show sd for this subject group. Section B plots the visual benefit (defined as the ratio of contrast sensitivities measured with and without correction of higher order aberrations in the eye), as a function of retinal illuminance. The visual benefit that follows correction of higher order aberrations is small when the retina is adapted to the corresponding ambient illumination. Replotted from Van Kvansakul, J., RodriguezCarmona, M., Edgar, D. F., et al. (2006). Supplementation with the carotenoids lutein or zeaxanthin improves human visual performance. Ophthalmic and Physiological Optics 26: 362–371; and Dalimier, E., Dainty, J. C., and Barbur, J. L. (2008). Effects of higher-order aberrations on contrast acuity as a function of light level. Journal of Modern Optics 55: 791–803.

Other features are also shared between the spatial and temporal CSFs. First, at low temporal frequencies, like low spatial frequencies, the contrast sensitivity becomes roughly constant and therefore independent of light level – in this case, at 0.65 phot. td and above. Second, as the retinal illuminance is increased, the highest temporal frequency that can just be seen (i.e., the temporal acuity limit, which is also known as the critical fusion frequency) increases from about 13 Hz at 0.06 phot. td to 80 Hz at 9300 phot. td.

The changes in the shapes of the temporal CSFs and the reduction in integration time both reflect a speeding up of the visual response with light adaptation. This speeding up can be assessed more directly by measuring the differences in visual delay between the two eyes when they are in different states of adaptation. Figure 8(a) shows how the cone response speeds up as the retinal illuminance in one eye is increased (relative to the cone

Figure 7 Temporal contrast sensitivity functions from 0.006 to 9300 td measured by Kelly (1962). Replotted from Kelly (1962). Visual responses to time-dependent stimuli I. Amplitude sensitivity measurements. Journal of the Optical Society of America 51: 422–429.

response in the other eye fixed at a retinal illuminance of 4.16 log phot. td). In terms of phase delay (where 360 is one cycle of flicker), the response speeds up, for example, at 10 Hz, by about 150 between 1.05 and 4.16 log phot. td. This is equal to 150/360 or 0.42 of a cycle, which, given that one 10-Hz cycle lasts 100 ms, represents a speeding up of roughly 42 ms. The visual response mediated by rods also speeds up over the scotopic range. Figure 8(b) shows the rod response in one eye with varying retinal illumination relative to the rod response in the other eye fixed at a retinal illuminance of 1.3 log scot. td. At 4 Hz, the response speeds up by 135 or 0.38 cycles between –3.3 and –0.8 log scot. td. This translates to a speeding up of roughly 94 ms. These results illustrate that changes in visual delay can be substantial. Visual performance, in terms of the speed of the visual response, improves markedly as the rod or the cone systems light adapt.

At mesopic levels, the situation is more complicated. Although the rod system is relatively light adapted and the cone system relatively unadapted at these levels, the rod system is still more sluggish than the cone system. These differences reflect intrinsic differences between the speeds of the responses of the rod and cone photoreceptors, as well as differences between the rod and cone postreceptoral pathways. The situation is further complicated by an abrupt transition that occurs within the rod system at mesopic levels from a slow, sensitive postreceptoral pathway to a faster, more insensitive one. Due to the complex differences between the temporal properties of rodand cone-mediated vision, mesopic measures of luminous efficiency and visual performance will be strongly dependent not only on the relative sensitivities

Levels (log phot. td)

Frequency (Hz)

Figure 8 Binocular phase-delay measurements made between the two eyes in different states of adaptation.

(a) M-cone phase delays in degrees between signals generated in the left eye and those generated in the right eye for observer ML. The adaptation level in the right eye was fixed at 4.16 log phot. td; that in the left eye was varied according to the key. Data replotted from Figure 5 of Stockman, A., Langendo¨rfer, M., Smithson, H. E., and Sharpe, L. T. (2006). Human cone light adaptation: From behavioral measurements to molecular mechanisms. Journal of Vision 6: 1194–1213. (b) Rod phase delays degrees between signals generated in the left eye relative and those generated in the right eye for observer LTS. The adaptation level in the right eye was fixed at 1.30 log scot. td; that in the left eye was varied according to the key. Data replotted from Figure 9 of Stockman and Sharpe (2006). Into the twilight zone: The complexities of mesopic vision and luminous efficiency. Ophthalmic and Physiological Optics 26: 225–239.

of the rods and cones, but also on the temporal characteristics of the stimuli used to make those measurements. Thus, targets of different duration are likely to produce mesopic results with different rod to cone weightings.

Color Vision and Light Level

Color signals contribute significantly to the detection and appearance of objects, particularly when the photopic

luminance contrast of the stimulus is low. In fact, in terms of cone-contrast signals under optimal conditions, the eye sees color better than it sees luminance. Other studies also show that even the pupil of the eye responds more vigorously to chromatic than to luminance signals when small stimuli are involved. Color can be perceived at or near the cone-detection threshold. For instance, the appearance of a spectrally green target is achromatic at scotopic levels, but takes on a clearly green tinge at mesopic levels. Color affects the conspicuity of stimuli. In general, the effective contrast of visual stimuli can be expressed as a complex function of photopic and scotopic luminance contrast with significant contributions from red/green and yellow/blue color signals in the photopic range. Importantly, chromatic signals also contribute to the perception of brightness (but not to luminance). In the mesopic range, cone signals become less effective, but the stronger rod signals do not appear to contribute significantly to color vision, particularly when threshold measurements are involved. When the stimulus is well above threshold, rod signals affect both the color appearance and the overall conspicuity of the stimulus.

Conclusions

The idea that the performance of the visual system can be usefully characterized over a 10 000-million-fold range of light levels by just three spectral luminous efficiency functions, corresponding to the scotopic, mesopic, and photopic ranges, is overly ambitious. Any model based solely on spectral sensitivity is unlikely to capture the effects of the large number of parameters that can influence object appearance. Here, we have focused on the way in which other key aspects of visual performance, such as spatial and temporal contrast sensitivity and acuity, visual delay, and color sensitivity, change with light level. We argue that only by linking such changes with changes in spectral luminous efficiency could we reasonably hope to predict visual performance, particularly at mesopic levels where the characteristics of the rod and cone systems are so different.

See also: Acuity; Chromatic Function of the Cones; Color Blindness: Acquired; Color Blindness: Inherited; Contrast Sensitivity; Information Processing: Contrast Sensitivity; Information Processing: Retinal Adaptation; Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Inactivation in Cones; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Phototransduction: The Visual Cycle.

Further Reading

Chisholm, C. M., Evans, A. D., Harlow, J. A., and Barbur, J. L. (2003). New test to assess pilot’s vision following refractive surgery. Aviation, Space, and Environmental Medicine 74(5): 551–559.

Dalimier, E., Dainty, J. C., and Barbur, J. L. (2008). Effects of higherorder aberrations on contrast acuity as a function of light level.

Journal of Modern Optics 55: 791–803.

Kelly, D. H. (1961). Visual responses to time-dependent stimuli I. Amplitude sensitivity measurements. Journal of the Optical Society of America 51: 422–429.

Kvansakul, J., Rodriguez-Carmona, M., Edgar, D. F., et al. (2006). Supplementation with the carotenoids lutein or zeaxanthin improves human visual performance. Ophthalmic and Physiological Optics 26: 362–371.

Sharpe, L. T. and Stockman, A. (1999). Two rod pathways: The importance of seeing nothing. Trends in Neurosciences 22: 497–504.

Stockman, A., Ja¨gle, H., Pirzer, M., and Sharpe, L. T. (2008). The dependence of luminous efficiency on chromatic adaptation. Journal of Vision 8(16): 1–26.

Stockman, A., Langendo¨rfer, M., Smithson, H. E., and Sharpe, L. T. (2006). Human cone light adaptation: From behavioral measurements to molecular mechanisms. Journal of Vision 6: 1194–1213.

Stockman, A. and Sharpe, L. T. (2006). Into the twilight zone: The complexities of mesopic vision and luminous efficiency.

Ophthalmic and Physiological Optics 26: 225–239.

van Nes, F. L. and Bouman, M. A. (1967). Spatial modulation transfer in the human eye. Journal of the Optical Society of America 57: 401–406.

Walkey, H. C. and Barbur, J. L. (guest editorial) (2006). Shedding new light on the twilight zone. Ophthalmic and Physiological Optics

26: 223–224.

Walkey, H. C., Barbur, J. L., Harlow, A., and Makous, W. (2001). Measurements of chromatic sensitivity in the mesopic range. Color Research and Application 26: 36–42.

Walkey, H. C., Barbur, J. L., Harlow, J. A., et al. (2005). Effective contrast of colored stimuli in the mesopic range: A metric for perceived contrast based on achromatic luminance contrast. Journal of the Optical Society of America 22: 17–28.

Glossary

Cell competence – In the context of retinal development, it refers to the characteristic ability of retinal progenitor cells (RPCs) to generate particular retinal cell types. It is determined by a combination of intrinsic and extrinsic factors.

Cell-fate determination – An early step (generally irreversible) in the developmental process leading to the acquisition of a differentiated cellular phenotype.

Cell-fate specification – Developmental process leading to the acquisition of a reversible cell fate, generally preceding determination.

Conditional knockout mouse – A genetically engineered mouse in which targeted deletion of gene(s) of interest can be temporally and/or spatially controlled.

Knockout mouse – A genetically engineered mouse carrying targeted deletion of one or more genes that is transmitted through the germ-line. Knockout mice in which gene function is completely ablated are referred to as null for that gene.

Phototaxis – Movement in response to light stimuli. Precursor – For the purposes of this article, and in agreement with previously published material, the term precursor is used for a postmitotic cell committed to a specific cell fate but not having the differentiated cell function or phenotype. Other authors may use the term precursor interchangeably with the term progenitor.

Progenitor – For the purposes of this article and in agreement with previously presented material, a progenitor is a proliferating, undifferentiated, multipotent, yet developmentally restricted, cell that expresses a combination of genes biasing its fate toward one or multiple differentiated retinal phenotypes.

Rhabdomere – A visual pigment-containing organelle, characterized by numerous microvilli, normally found on the apical surface of photoreceptors, mostly in arthropods. Its counterpart in vertebrates is the outer segment of rods and cones. Transgenic mouse – A mouse genetically engineered with random integration of one or more exogenous genes (transgenes) in its genome. Expression of the transgene can be cell/tissue specific and controlled over time.

Introduction

Rod and cone photoreceptors are highly specialized sensory neurons responsible for the detection of visual stimuli. They convert quanta of light into signals that are transmitted via interneurons (bipolar cells) to projection neurons (retinal ganglion cells (RGCs)) and then to the brain, where neuronal electrical stimuli are interpreted as visual sensation. In vertebrates, rod and cone photoreceptors are characterized by the expression of rhodopsin and cone opsins, respectively. Opsins belong to a family of membrane-bound guanine nucleotide binding proteincoupled receptors, which are covalently linked to a vitamin A-derived retinaldehyde chromophore and are responsible for initiating the phototransduction cascade.

The capacity to detect and respond to changes in environmental illumination is a fundamental survival skill present, to different degrees, in all living beings. Opsinlike molecules first appeared in prokaryotes (bacteria and archaea) and could function as proton-pumps to transfer energy within a unicellular organism. Activation of opsinlike molecules in response to circadian rhythms and dark–light cycles might sustain a primitive form of flagella and pili phototaxis. The capacity to perceive light stimuli further evolved in protozoa, where opsins allow for rudimental vision to sense the environment and guide adaptive movements. However, only metazoans (animals) display anatomical structures dedicated to detection and processing of light stimuli, with graded complexity from the pit eyes of worms and mollusks to the camera-type eyes of vertebrates, all of which contain light-responsive photoreceptors.

Evolution has resulted in two primary types of photoreceptor cells: rhabdomeric and ciliated. In both, the cell membrane has evolved to accommodate the maximum number of opsin molecules to increase the probability that one is activated by a single photon of light. Photoreceptors in most invertebrates are rhabdomeric with thousands of opsin-containing microvilli, whereas most vertebrates have a ciliated structure containing multiple invaginations of opsin-rich stack of membranous disks, known as the outer segment. Furthermore, as described elsewhere in this encyclopedia, other marine mollusks – such as the Pectin – contain rhabdomeric and ciliary photoreceptors in separate layers in the same retina. While phototransduction leads to membrane depolarization in rhabdomeric photoreceptors, it results in hyperpolarization in ciliated photoreceptors. Developmental

processes also differ between invertebrates and vertebrates, even in those species displaying a camera-type structure. In the former (e.g., the octopus), the eye is of epidermal origin and mature photoreceptors face the vitreous cavity. In the latter, the eye develops from the neural plate (neuroectodermal origin) and mature photoreceptors are oriented away from the vitreous.

This article follows the developmental pathway that results in the generation of rods and cones from pools of retinal progenitor cells (RPCs) in the mammalian retina. We describe the factors involved in photoreceptor cellfate specification, determination, and maturation. We have focused on human photoreceptors for their relevance in retinal degenerative diseases and on the mouse because of abundant available literature in this model pertaining to photoreceptor development.

Photoreceptor Development

Rod and Cone Pattern in Human and

Mouse Retina

Photoreceptors are characterized by distinct morphology and synaptic connections that reflect their unique functions in light detection and visual process. Rod photoreceptors function in dim light, whereas cones are responsible for chromatic vision and visual acuity in bright light. These differences in light sensitivity are made possible by distinct visual pigments (opsins) and other proteins that mediate the phototransduction cascade.

All rod photoreceptors express rhodopsin from the OPN2/RP4/RHO gene and display peak sensitivity at a wavelength of approximately 500 nm. Cone photoreceptors can be further distinguished in different subtypes, based on the wavelength sensitivity conferred by their characteristic opsin photopigment. Humans and old world primates have three subtypes of cone photoreceptors. Cones sensitive to blue light express short-wavelength- sensitive (S)-opsin from the OPN1SW gene (S cones); cones sensitive to green light express medium-wavelength- sensitive (M)-opsin from the OPN1MW2 gene (M cones); whereas those sensitive to red light express long wavelength-sensitive (L)-opsin from the OPN1LW gene (L cones). On the other hand, only S-opsin and M-opsin from Opn1sw and Opn1mw genes, respectively, are expressed in mouse cone photoreceptors.

In humans and mice, rods represent over 95% of the photoreceptors. The remaining 5% (human) and 3% (mouse) are cone photoreceptor subtypes. Rods and cones are arranged in a spatial mosaic in the human retina, where cones are concentrated in a region at the center of the macula – the fovea. The innermost 100-mm-wide central fovea – known as the pure cone area – contains a dense population of L and M cones and it is completely devoid of S cones, which are distributed in the peripheral fovea. Rods

appear only approximately 300 mm from the center of the fovea and their density peaks outside the fovea at the eccentricity of the optic disc. A smaller proportion of cones are found interspersed within the more numerous rods in the peripheral retina. The ratio of L-to-M cones in the pure cone area and between central and peripheral retina varies considerably among individuals, and no characteristic cone-subtype distribution pattern is evident.

In the mouse retina, rods are relatively homogeneously distributed, and there is no structure resembling the human fovea. However, S- and M-opsins are expressed in cones in a reciprocal dorsal-to-ventral gradient with S-opsin-expressing cones localized predominantly in the ventral portion of the retina and M-opsin cones predominantly in the dorsal region. Differences in photoreceptor subtypes and distribution are reflected in the developmental events that generate photoreceptors in the human and mouse retina.

Development of Cone and Rod Photoreceptors

The retinal neuroepithelium is populated by proliferating, undifferentiated, multipotent RPCs (neuroblasts), from which all neural retinal cell types – including rod and cone photoreceptors – are generated during retinogenesis. Cell birth refers to the time when a cell exits the final mitotic cycle and commits to a specific differentiated fate. Birth-dating studies in the murine retina have shown that RPCs destined to become cone photoreceptors exit the cell cycle between embryonic day (E) 11 and E18 starting from the central retina and proceeding toward the periphery (Figure 1(a)). In an overlapping wave, rod progenitors exit the cell cycle between E12 and postnatal day (P) 10. Initiation of opsin protein expression occurs later, with S-opsin protein first detected around E19, followed by rhodopsin around P2 and M-opsin around P7.

Immunohistochemical analysis of the developing human retina, together with data from Macaque monkeys, suggests that cone photoreceptors are generated in the human retina starting from the prospective fovea in a period of time from fetal week (Fw) 8 to Fw34 (Figure 1(b)). In humans, rod genesis begins after cone birth around Fw10 but overlaps with cone genesis and continues into the first 8 months of postnatal life. Unlike the mouse, S-opsin expression in humans initiates around Fw12, preceding rhodopsin and L-/M-ospin expression, which are evident by Fw15. In humans, synaptic and neurotransmitter proteins are expressed in association with the initiation of synaptogenesis during the lag period between cone genesis and production of cone opsin. However, synaptogenesis in rods occurs only after the onset of rhodopsin synthesis.

In the mouse, the above-mentioned events occur first in the center of the retina and proceed toward the periphery; this is similar to humans where, generally, new developmental events start in the pure cone area. In the early postnatal human retina, the fovea is still immature with a

Figure 1 Photoreceptor development in mouse (a) and human

(b) retina. In the first stage of photoreceptor development, prospective cone and rod precursors exit the cell cycle in sequential (cone first and rod after), yet overlapping waves (dotted lines). Postmitotic cells lose their apical process while retaining their connection to the outer limiting membrane and have elongated cuboidal morphology (cones) or a round shape (rods). The amplitude of the dotted curves in (a) and (b) represents the proportion of cells becoming postmitotic and fated to differentiate into cones (purple) and rods (orange), and the period of time in which all cells exit the cell cycle. Synthesis of specific opsins initiates the differentiation stage. Solid lines in (a) and (b) illustrate the temporal protein expression pattern of rhodopsin (brown), S-opsin (blue), M-opsin (green), and L-/M-opsin (red). In the mouse (a), S-opsin (blue) is expressed first, followed by rhodopsin (brown) and then M-opsin (green). The relative amount of rhodopsin expressed is considerably higher than S- and M-opsin, consistent with the higher proportion of rods (>95%) in the photoreceptor cell population. Photoreceptor maturation is completed between the second and the third postnatal week (arrow). In the human (b), S-opsin is synthesized first, followed shortly after by overlapping rhodopsin and L-/M-opsins. Rhodopsin production increases dramatically after birth, coincidental with outer segment formation and maturation. Cone and rod photoreceptors are rearranged after birth, with packing of cones in the fovea (dashed arrow). Outer segments continue to expand until they reach their mature size by the third year of age (solid arrow). B, birth; OS, outer segment.

greater proportion of rods than cones at the edge of the pure cone area. The mature fovea becomes evident by 1 year of age with the characteristic packaging of cones in the center. Rod outer segments continue to grow in length well into the first postnatal years until the human retina reaches full maturation by approximately 3–5 years of age.

Factors Affecting Photoreceptor Genesis

In the stereotypical progression of cell differentiation in the retina, cone genesis is initiated from common proliferating RPCs, in a sequential manner after RGCs and before horizontal and amacrine neurons. Rod birth is initiated later compared to cone genesis, but it precedes bipolar and Mu¨ller glia cells. However, the time intervals during which full complements of cone and rod photoreceptors are generated largely overlap (see Figure 1). Lineage and birth-dating analyses of mouse retina indicate that photoreceptor cell-fate decisions are made at the time of terminal mitosis; however, additional investigations are necessary. Two key elements control the commitment and differentiation of photoreceptors: gene-regulatory networks that confer competence to the RPC and dictate differentiation events (Figures 2 and 3), and extrinsic factors that modulate transcriptional cascades in differentiating RPCs and later modulate cell–cell communication (Figure 2). In addition, epigenetic mechanisms (such as chromatin modifications) appear to play a significant role in directing photoreceptor specification and differentiation.

Early Stages in Photoreceptor

Development

From RPC to Photoreceptor Precursor

At the time of their last mitosis and prior to exiting the cell cycle, RPCs fated to become photoreceptors upregulate the expression of paired-class homeodomain transcription factor orthodenticle protein homolog 2 (OTX2) (Figure 2). In the mouse, OTX2 is first detected at E11.5–12.5 in the retinal neuroblastic layer (Figure 3). OTX2 expression increases thereafter, concomitantly with early cone and rod development, and persists in the postnatal mouse retina in bipolar and photoreceptor cells. Otx2 plays an important role as an early factor that specifies photoreceptor cell fate, and probably as a late factor that may promote terminal differentiation by participating in upregulation of photoreceptor-specific genes. When Otx2 is conditionally knocked out in the developing mouse eye, photoreceptors do not develop and are replaced by amacrine-like cells. Furthermore, when Otx2 is ubiquitously expressed in RPCs, all cells follow the photoreceptor fate. In humans, OTX2

S-cone precursor

RHO+

Arrestin+

PDE6+

NRL+

Rod

Mopsin+

CAR+

TRβ2+

RXRγ+

M-cone

S-opsin+

CAR+

TRβ2−

RXRγ−

S-cone

mutations are associated with retinal diseases, such as anophthalmia, microphthalmia, and coloboma.

At the molecular level, OTX2 is shown to activate the promoter of cone–rod homeobox (CRX) transcription factor, a closely related homeodomain transcription factor. The two transcription factors (OTX2 and CRX) appear to promote completion of photoreceptor differentiation programs (Figure 2). CRX does not contribute to photoreceptor cell specification, but is essential for terminal differentiation and maintenance. In Crx-null mice, despite normal photoreceptor genesis, morphogenesis is incomplete as outer segments fail to elongate, and photoreceptors do not produce a full complement of phototransduction proteins. These abnormal photoreceptors undergo synaptogenesis, but their synaptic endings appear malformed and eventually degenerate. Mutations in the human CRX result in retinopathies, including Leber

congenital amaurosis (LCA), cone–rod dystrophy, and retinitis pigmentosa (RP).

Another protein suggested to participate in photoreceptor cell development is the basic helix-loop-helix (bHLH) transcription factor NeuroD1 (Figure 2). NeuroD1 is expressed in developing and differentiated rod and cone photoreceptors. Its targeted deletion in the mouse, however, causes only a modest decrease in photoreceptor number. NeuroD1 acts in combination with another bHLH transcription factor, MASH1, which may contribute to regulating the timing of photoreceptor differentiation as its targeted deletion in mouse leads to delay in photoreceptor development.

Very early, at the time of photoreceptor specification, postmitotic precursor cells become committed toward rod or cone fates. The Maf family basic motif-leucine zipper transcription factor NRL and its target, the

(d)

NBL

GCL

(b)

Rod photoreceptor

*

P2-6

photoreceptor-specific orphan nuclear receptor NR2E3, are the primary determinants of rod versus cone cell-fate determination. It appears that S cone cell fate is specified as a default pathway and that inhibition of this pathway by NRL/NR2E3 is permissive to rod specification (Figure 2).

From Cone Precursor to Cone

Photoreceptor

Cones are the first photoreceptor cell type to exit the cell cycle in all vertebrates (Figure 1). However, their differentiation takes a few days (mouse) to weeks (human) and is complete only after a majority of rods have become postmitotic. Cone precursors must go through an additional specification process that determines distinct cone subtypes (L, M, or S in humans, and M or S in mouse).

Studies of cone-photoreceptor-fate determination rely 8w?>on the detection of specific opsins at the mRNA or protein level, thus overlapping with studies on opsin gene

regulation. In mouse and human cone precursors, S-opsin expression is detected first, followed by M-opsin expression (Figure 1). Variable numbers of photoreceptors go through a transitional state in which both opsins are expressed, before S-opsin is downregulated and M-opsin predominates. In the adult mouse retina, a prevalent population of photoreceptors coexpresses both pigments, whereas cone photoreceptors in the human retina express a single opsin.

In mice, onset of cone opsin expression is regulated by numerous factors; these include CRX, members of the nuclear receptor family, thyroid hormone nuclear receptor beta 2 isoform (TRb2) and retinoid X nuclear receptor gamma (RXRg), and of the retinoic acid (RA) receptorrelated orphan receptor – RORa and RORb2. RORa and RORb2 are expressed in postmitotic cone-photoreceptor precursors and directly regulate S-opsin expression synergistically with CRX. Furthermore, RORa also appears to regulate the expression of M-opsin through direct binding to its promoter, highlighting a potentially more complex role in cone differentiation. Notably, Rorb2-null

mice lack outer segments, suggesting RORb2 plays an additional role in promoting photoreceptor maturation.

TRb2 and RXRg are also expressed in postmitotic cone precursors in the neonatal mouse retina (Figures 2 and 3) and act as transcriptional repressors of S-opsin expression. In fact, both receptors are downregulated concomitant with the initial onset of S-opsin expression and upregulated at a later stage in M cones in the dorsal retina to suppress S-opsin. Further evidence comes from studies of Rxrg-null and Trb2-null mice. Both nuclear receptors are necessary to repress S-opsin expression and probably act in concert on the S-opsin promoter. However, only TRb2 is required for M-opsin expression, making it a key regulator of M cone differentiation. Trb2 gene appears to be regulated by a transcriptional complex containing NeuroD1. Although NeuroD1 alone is not sufficient to initiate Trb2 expression, it is required for sustained gene activation, thus supporting its role in M cone differentiation.

To date, there is no evidence involving RA in cone development in the mouse or human. On the other hand, TH has been suggested to regulate the ratio and patterning of cone-photoreceptor subtypes through changes in geographical distribution in the developing retina.

From Rod Precursor to Rod

Photoreceptor

NRL is the master regulator of rod cell fate and serves as the unique defining signature of newborn rod photoreceptors (see Figures 2 and 3). NRL induces the expression of NR2E3. Following this, NRL and NR2E3 – together with CRX – lead to the expression pattern typical of mature rod photoreceptors (Figure 2). In mice null for the Nrl gene, all rods are converted to cones. Targeted deletion of Nr2e3 in the mouse leads to enhanced S-cones with hybrid photoreceptors. Transgenic expression of Nr2e3 in Nrl-null mice suppresses cone differentiation and leads to the generation of rod-like photoreceptors, yet NR2E3 is not sufficient to produce functional rods. These data support a model in which active induction by NRL and NR2E3 in CRXexpressing photoreceptor precursors is required for rod cell-fate determination with simultaneous inhibition of the cone pathway (Figure 2).

Mutations in NRL are associated with retinal degenerative diseases, including autosomal dominant and recessive RP, and retinopathies with varying phenotypes. Loss of NR2E3 in humans leads to enhanced S-cone syndrome, Goldmann–Favre syndrome, and similar retinopathies with increased S-cone function.

The retinoblastoma (Rb) gene appears to be an important intrinsic regulator of rod development. In the postnatal mouse retina, Rb is expressed in mitotic RPCs, where it regulates timely exit from the cell cycle, and in

differentiating rod photoreceptors. When Rb is deleted, RPCs continue to proliferate and rod photoreceptors do not develop. Unlike Nrland Nr2e3-null mice, rod photoreceptors in Rb-null mice do not change their fate to cones (cone number remains unmodified). Rather, their development is arrested at the progenitor stage. It remains unclear from the current literature whether Rb is instructive or permissive for rod-photoreceptor cell fate.

Several extrinsic factors contribute to signaling for rod development (Figure 2). Cell–cell interaction mediated by Notch1 is known to sustain the undifferentiated and proliferating state of RPCs, repressing neuronal fate in general. Recently, Notch1 signaling has been shown to specifically inhibit photoreceptor fate, that is, cone in the embryonic and rod in the postnatal mouse retina. This function of Notch1 may allow differentiation of other neuronal cell types as light detection evolved from uniquely photoreceptor-based, in lower species, to multiple cell-type-mediated, in higher species. Other extrinsic factors inhibiting rod fate are leukocyte inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), and epidermal growth factor (EGF).

Taurine, secreted by RGCs, is suggested to stimulate rod photoreceptor production through glycine (Gly) and gamma aminobutyric acid (GABA(A)) receptors. The hypothesis that taurine and its receptor GlyRa2 have a role in promoting rod cell fate awaits further validation.

Proper maturation and/or migration and integration of young rods into the outer nuclear layer (ONL) may require gradients of Indian Hedgehog (IHH) secreted by the retinal pigment epithelium (RPE), and Sonic Hedgehog (SHH) secreted by RGCs. IHH may also have a role in the specification of photoreceptor fate, possibly inducing Nrl in photoreceptor precursors. Similarly, fibroblast growth factor (FGF) family members – acidic FGF (FGF1) and basic FGF (FGF2) – are implicated in rod maturation and may induce NRL expression.

The role of RA in promoting rod photoreceptor differentiation is still unclear, though it can induce Nrl expression and RA-responsive sites are present in the Nrl promoter. However, retinoic acid receptor (RAR)b2/ RARg2 double null mutant mice contain a normal photoreceptor complement.

Activin A – a transforming growth factor beta (TGFb)- like protein expressed by extraocular mesenchyme and RPE – promotes photoreceptor development in vitro and in vivo, and increases the number of photoreceptor cells in rat retinal cultures. In vitro, it causes RPCs to exit the cell cycle and biases them to become rods, but not cones. In vivo, mice with homozygous deletion of activinA show substantial decrease in the number of photoreceptors. Finally, growth differentiation factor 11 (GDF11) and related TGFb family members control the competence of mitotic progenitors to acquire rod cell fate. GDF11-null retinas have more RGCs at the expense of photoreceptors.

Maturation of Photoreceptors

Cones are generated in mice during the prenatal period. Rod photoreceptor birth overlaps with the genesis of all retinal cell types, though most rods are born postnatally (Figure 1). Maturation of committed precursors to differentiated functional photoreceptors is a lengthy process and involves expression of cell-type-specific phototransduction genes, biogenesis of outer segments, and formation of synapses with specific interneurons. Expression of most photoreceptor-enriched genes depends on the synergistic or antagonistic actions of NRL, NR2E3, and CRX and their interaction with other regulatory proteins. In most instances, these proteins co-occupy the promoter/ enhancer regions of their target genes. Mutations in the target genes of NRL and CRX are associated with retinal dysfunction.

Studies with transgenic and knockout mice, together with microarray and chromatin immunoprecipitation analysis, have yielded valuable information about gene-regulatory networks that guide photoreceptor differentiation and maturation. As NRL and its direct target nuclear receptor NR2E3 determine rod cell fate, these two transcription factors activate the expression of rod-specific genes and repress cone-gene expression. NRL, even in the absence of NR2E3, can activate all rod-specific genes (e.g., rhodopsin, PDE-a, PDE-b) but the repression of cone genes (e.g., S-opsin) is not efficient, leading to hybrid photoreceptors in mice expressing NRL but not NR2E3 (rd7 mice). NR2E3, on the contrary, can repress cone genes but it is unable to efficiently activate rod genes in the absence of NRL. Both NRL and NR2E3 interact with a multitude of regulatory proteins to accomplish transcriptional regulation. NRL is a highly phosphorylated protein and its function is modulated by several kinases. NRL also interacts with TATA-binding protein (TBP) and, presumably, brings the basal transcriptional machinery to target gene promoters. Recent studies have shown a key role for the protein inhibitor of activated STAT3 (PIAS-3) in the sumoylation of NR2E3, adding another level of control in gene expression and consequently rod differentiation. A combined action of NRL and NR2E3 is essential to generate functional rod photoreceptors.

CRX, on the other hand, acts as an enhancer of both rod and cone genes. While photoreceptors are produced even in the absence of CRX, these cells do not elaborate outer segments because of the low expression of most, if not all, phototransduction and structural proteins. CRX is, therefore, necessary to produce functional photoreceptors. CRX also interacts with coactivator proteins that possess histone acetyltransferase (HAT) activity and recruits HATs to promoter/enhancer regions to acetylate histone H3, thereby inducing and maintaining chromatin configurations that facilitate binding of NRL, NR2E3, and RNA

polymerase II. These recently described molecular mechanisms underscore the importance of as yet poorly understood epigenetic factors in determining retinal photoreceptor cell fate and maturation.

Current Research in Photoreceptor

Development

Cell interactions and intrinsic cellular mechanisms modulate gene expression and regulate photoreceptor differentiation. Among these, chromatin remodeling and small regulatory RNAs have attracted attention in recent years.

Histone methylation/acetylation are key epigenetic modifications that govern chromatin dynamics. The role of chromatin-modifying activities in directing tissue-specific development is an active area of investigation. Histone acetylation is emerging as an important mechanism in regulating photoreceptor development. Acetylation of histone H3 by HATs – recruited by CRX – appears important in maintaining chromatin configurations permissive to NRL/NR2E3 transcriptional activity in developing rods. Furthermore, histone deacetylase 4 (HDAC4) activity is shown to promote the survival of newly differentiated photoreceptors. More recently, the chromatin-remodeling complex Baf60c has been identified in differentiating, but not mature, retinal cells.

A family of three DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, partially cooperate to establish and maintain genomic DNA methylation patterns. The presence of high levels of Dnmt3a and of Dnmt3b in the mouse rostral neural tube, including the optic grooves and cranial neural folds at E8.5, and in the area of evaginating optic vesicles at E9.5 is suggestive of the role Dnmts play in eye development. Epigenetic chromatin-remodeling mechanisms are also active in retinal neuroblasts undergoing cell-fate commitment, and are likely to contribute to retina-restricted patterns of gene expression. For example, hypomethylation is suggested to modulate interphotoreceptor retinoid-binding protein (IRBP) gene activation during photoreceptor genesis. Little is known about the influence of methylation on retinal cell fate, yet it is plausible that DNA methylation is actively involved in establishing RPC competence.

MicroRNAs (miRs) are short (18–24 nucleotides), noncoding, RNA sequences that modulate gene expression by binding the 30 (and 50 ) untranslated region (UTR) of their target RNAs, thus regulating their stability and translation. They originate as longer RNA transcripts that are processed and cleaved by the subsequent activity of two RNase III endonucleases – the Drosha-DGCR8 complex and Dicer. MiRs could play a gene-regulatory role in development (including retinal) that is comparable to that of transcription factors. For example, in the Drosophila eye,