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

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retinal area. However, VEGF mRNA is not detectable in the human retina until 20 WG. Formation of the inner plexus is well underway by 14–15 WG, prior to the differentiation of most retinal neurons. Finally, the development

of the inner plexus is centered at the optic disk, whereas neuronal maturation is centered at the fovea. In contrast, development of the outer plexus by angiogenesis is centered on the fovea.

Angiogenic and anti-angiogenic factors

Retinal angiogenesis is controlled by a balance of proangiogenic and antiangiogenic factors. VEGF is a pro-angiogenic factor that has several forms: VEGFA (there are four isoforms, including VEGFA165), VEGFB, VEGFC, and VEGFD. There are at least three VEGF receptors: VEGFR1, 2, and 3. VEGFA is not only a proangiogenic factor, but it also has protective functions. Further, it prevents vessel regression and appears to stabilize the mature vasculature, rendering mature vessels resistant to fluctuations in tissue oxygen levels. VEGFA also has a protective effect in the maintenance and function of the neuronal and Muller cells of the retina. VEGFR3 is required for normal retinal vascular development and is upregulated during VEGFA-induced angiogenesis. Its ligand, VEGFC, can cause blood vessel enlargement and tortuosity, and in vitro has a synergistic effect on angiogenesis with VEGFA.

Anti-angiogenic factors in the retina include PEDF and vascular endothelial growth inhibitor (VEGI). PEDF is synthesized by cells of the retinal pigment epithelium (RPE), and is present in the developing embryonic and adult human RPE, choroid, and retina. In the human, it is unclear as to how the expression of PEDF relates to the developing vasculature. VEGF stimulates PEDF expression by RPE cells but suppresses PEDF expression by Mu¨ller cells. VEGI inhibits endothelial cell proliferation and causes the apoptosis of proliferating endothelial cells. It is expressed by endothelial cells and is thought to play a role in the maintenance of a quiescent adult vasculature. Little is known about VEGI expression in ocular tissues. VEGI192 may not be expressed during retinal and choroidal vessel formation but instead may be expressed by the endothelial cells of the quiescent adult vasculature, and may be associated with mural cell maturation. The relative expression of proand antiangiogenic growth factors ultimately determines whether neovascularization takes place once a stable quiescent vascular plexus is reached in young adulthood.

Cell–Cell Interactions in the Formation

of the Human Retinal Vasculature

Mu¨ller cell–endothelial cell interactions

Along with astrocytes, Mu¨ller cells are found surrounding the vessels of the inner plexus of the retina, and are thought to be involved in the establishment of the BRB. However, astrocytes are not found in the deeper plexus of the retina; these vessels are ensheathed solely by Mu¨ller cells. The existence of an intact BRB and the presence of tight junction proteins in the deep plexus suggest that in this layer of vasculature, Mu¨ller cells are capable of inducing the formation and maintenance of barrier properties.

Pericyte–endothelial cell interactions: Vessel stability

Pericytes are another integral component of the mature retinal vasculature. These cells are found within the basal lamina that ensheaths capillary-sized vessels, and play a role in vessel stabilization, control of blood flow, and possibly in modulating vascular permeability and maintenance of the BRB. Pericyte density in the retina is the highest of any tissue, and these cells are also the first to be lost in diabetic retinopathy. Although immature pericytes associate with newly formed retinal vessels (Figure 7(c) and 7(d)), the presence of these immature mural cells does not prevent vessel regression. However, mature vessels that are ensheathed by mature pericytes are quite stable and resistant to VEGF165 withdrawal, suggesting that pericytes must be mature and fully differentiated to contribute to vessel stability. A reciprocal relationship exists between endothelial cells and pericytes; whereas pericytes express VEGF165 and contribute to vessel stability, retinal endothelial cells express PDGFB, which induces recruitment and proliferation of pericytes.

Pericytes and smooth muscle cells constitute the mural cells, an important component in the maturation and stabilization of vessels. Mural cells are recruited to ensheath immature endothelial tubes by the localized expression of platelet-derived growth factor (PDGF). The inappropriate expression of PDGF interferes with the recruitment of mural cells and leads to abnormal retinal vascular remodeling. More specifically, the endothelial tube must be ensheathed by three components for a new vessel to become patent and stable: mural cells, a basal lamina, and astrocytes. Without any one of these components, vessels are leaky and unstable. Although the presence of immature pericytes along vessels does not protect these vessels from hyperoxia-induced regression, a mature vasculature with mature mural cells, astrocytes, and basal lamina is stable and resistant to the effects of hyperoxia. It appears that the extent of desmin expression by pericytes ensheathing immature vessels imparts upon its associated vascular segment vascular stability.

Vascular Remodeling

With the formation of an exuberant capillary plexus (Figure 3(c)) coupled with decreased metabolic demand as endothelial cell growth slows, the newly vascularized region becomes hyperoxic. Hyperoxia in turn leads to vascular remodeling, a paring back of the new vascular network to more precisely meet the metabolic needs of the underlying retina. Remodeling of the vascular network of the retina includes two processes: coalescence of smaller vessels into larger vessels and the retraction of endothelial cells from vessels that are pruned back (Figure 7(e)), followed by redeployment of most of these cells to areas where the vascular network is actively

forming. Most noticeably, capillaries retract near the arteries, forming a capillary-free space in this region (Figures 6(d) and 7(f )). Although some apoptosis is evident in the retracting vessels, cell death is not the main mechanism for vessel retraction. As the vascular network is remodeled and the metabolic demands of the tissue stabilize, the capillary network becomes very well organized and regular in appearance (Figure 7(g)). The process of vascular remodeling occurs over an extended period of time during human embryonic development.

Vascularization and the Health of the Eye

Interruptions to the normal cellular and molecular mechanisms of retinal vascular formation/function can lead to blindness. In infants, retinopathy of prematurity (ROP) develops when premature delivery and supplemental oxygen exposes an infant’s developing retina to elevated oxygen levels. This hyperoxygenation of the retina leads to a loss of the normal molecular cues that drives vessel formation such that when the infant is returned to room air, the rate of retinal vascularization is significantly delayed relative to neuronal maturation. A subsequent return to normoxic conditions generates retinal hypoxia that exceeds the normal physiologic levels, triggering the formation of leaky, tortuous vessels. This abnormal vasculature can lead to intraretinal hemorrhage and partial or complete retinal detachment.

In adults, the effects of tissue hypoxia and hyperglycemia can be seen in diabetic retinopathy. In this disease, the physiologic processes of diabetes prevent the retinal vasculature from meeting the metabolic demands of the active retina and generate locally high levels of hypoxia, in addition to the damaging effects of prolonged hyperglycemia. This retinal hypoxia stimulates angiogenesis in the context of a mature, fully developed retinal vasculature. Subsequent retinal neovascularization produces tortuous, fragile vessels over the surface of the retina. These vessels often leak fluid into the vitreous and can cause macular edema. In addition, the development of fibrous tissue in association with these vessels can lead to traction retinal detachments or vitreous hemorrhages.

Conclusions

The human retina is vascularized by a combination of two mechanisms: vasculogenesis and angiogenesis. Vasculogenesis is responsible for the formation of the earliest vessels of the inner plexus, starting at the ONH and reaching the inner two-thirds of the retina. Between that boundary and the inner periphery of the retina, in the space between these early vessels, reaching down to the outer plexus, under the fovea and over the nerve bundles at the optic nerve,

the process of angiogenesis drives the formation of the remaining vasculature. The formation of these vessels is controlled by physiological hypoxia – a level of hypoxia that is well tolerated by the tissue but is insufficient to cause tissue damage. Physiological hypoxia is sufficient to stimulate VEGF production (mediated by hypoxia inducible factor-1a (HIF-1a)) by astrocytes and Mu¨ller cells and to direct endothelial growth where it is needed. Once this proliferation of new vessels becomes patent and directs blood flow to the developing retinal tissues, a modest level of hyperoxia then orchestrates vascular remodeling and retraction, a process that is still ongoing in a full-term neonate. The balance between physiological hypoxia and hyperoxia, vessel growth and vessel retraction, leads to the formation of a vasculature that precisely meets the metabolic demands of the human retina.

See also: Physiological Anatomy of the Retinal Vasculature.

Further Reading

Adamis, A. P., Aiello, L. P., and D’Amato, R. A. (1999). Angiogenesis and ophthalmic disease. Angiogenesis 3: 9–14.

Chan-Ling, T. (2006a). Glial, neuronal and vascular interactions in the mammalian retina. Progress in Retinal and Eye Research

13: 357–389.

Chan-Ling, T. (2006b). The blood retinal interface: Similarities and contrasts with the blood–brain interface. In: Dermietzel, R., Spray, D. C., and Nedergaard, M. (eds.) Blood–Brain Barriers – From Ontogeny to Artificial Interfaces, pp. 701–724. Weinheim: Wiley-VCH.

Chan-Ling, T., Gock, B., and Stone, J. (1995). The effect of oxygen on vasoformative cell division. Investigative Ophthalmology and Visual Science 36: 1201–1212.

Chan-Ling, T., Halasz, P., and Stone, J. (1990). Development of retinal vasculature in the cat: Processes and mechanisms. Current Eye Research 9: 459–476.

Chan-Ling, T., McLeod, D. S., Hughes, S., et al. (2004). Astrocyte-endothelial cell relationships during retinal vascular development. Investigative Ophthalmology and Visual Science 45: 2020–2030.

Dorrell, M. I., Aguilar, E., and Friedlander, M. (2002). Retinal vascular development is mediated by endothelial filopodia, a preexisting astrocytic template and specific R-cadherin adhesion. Investigative Ophthalmology and Visual Science 43: 3500–3510.

Dorrell, M. I. and Friedlander, M. (2006). Mechanisms of endothelial cell guidance and vascular patterning in the developing mouse retina.

Progress in Retinal and Eye Research 25: 277–295.

Flynn, J. T. and Chan-Ling, T. (2006). Retinopathy of prematurity: Two distinct mechanisms that underlie Zone 1 and Zone 2 disease.

American Journal of Ophthalmology 142: 46–59. Fruttinger, M. (2007). Development of the retinal vasculature.

Angiogenesis 10: 77–88.

Gerhardt, H., Gloding, M., Fruttinger, M., et al. (2003). VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. Journal of Cell Biology 161: 1163–1177.

Hughes, S. and Chan-Ling, T. (2000a). Roles of endothelial cell migration and apoptosis in vascular remodeling during development of the central nervous system. Microcirculation 7: 317–333.

Hughes, S., Yang, H., and Chan-Ling, T. (2000b). Vascularization of the human fetal retina: Roles of vasculogenesis and angiogenesis. Investigative Ophthalmology and Visual Science

41: 1217–1228.

Kozulin, P., Natoli, R., Madigan, M. C., O’Brien, K. M. B., and Provis, J. M. (2009). Gradients of Eph-A6 expression in primate retina suggest a role in definition of the foveal avascular area. Molecular Vision

15: 2649–2662.

Kozulin, P., Natoli, R., O’Brien, K. M. B., Madigan, M. C., and

Provis, J. M. (2009). Differential expression of anti-angiogenic factors and guidance genes in the developing macula. Molecular Vision 15: 45–59.

Lutty, G. A., Chan-Ling, T., Phelps, D. L., et al. (2006). Proceedings of the Third International Symposium on Retinopathy of Prematurity: An update on ROP from the lab to the nursery (November 2003, Anaheim, California). Molecular Vision 12: 532–580.

Stone, J., Itin, A., Alon, T., et al. (1995). Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor ( VEGF) expression by neuroglia. Journal of Neuroscience 15: 4738–4747.

Glossary

Agenesis – Failure of an organ to develop during embryonic growth and development. Anophthalmia – Absence of the eye as a result of a congenital malformation during development. Coloboma – Congenital malformation due to failure of fusion of optic fissure.

Hyperplasia – Abnormal increase in cell proliferation within an organ or tissue due to the disregulation of cell-cycle control mechanisms.

Induction – Change in cell fate resulting from an external signal (inducer). This signal secreted by a group of cells instructs target cells to acquire specific properties.

Invagination – Morphogenetic process leading to the coordinated inward folding of a cell layer forming a pocket on the surface.

Mesenchyme – Embryonic mesodermal connective tissue composed of loosely packed unspecialized cells characterized by the ability to migrate into other tissues.

Phenotype – Expression of a specific trait based on genetic and environmental influences. Prosencephalon – Anterior-most division of the developing vertebrate brain (forebrain) that further subdivides into the telencephalon and the diencephalon.

Embryology

Eye development starts at late gastrula stages with the specification of retinal precursor cells within the eye field in the anterior neural plate. At this stage, retinal progenitor cells occupy a medial position and are surrounded rostrally and laterally by telencephalic precursors and caudally and medially by cells that will form the diencephalon. During neurulation, coordinated rearrangement of the cells that form each of these presumptive regions results in a new cell organization, whereby retinal progenitor cells now become laterally positioned. In vivo monitoring of retinal progenitor cell movement has shown that, at least in fish, dorsal diencephalic cells – positioned caudally to the eye field – move inward and forward,

displacing prospective eye and optic stalk tissue forward and then sidewise. At the same time, laterally positioned telencephalic precursors fold toward the midline leaving retinal precursors cells to the side of the neural tube (Figure 1). As these movements happen, the cells of the left and right eye, which were initially intermingled, segregate in their respective domains forming two visible optic vesicles.

At the present time, it is not clear whether similar morphogenetic movements occur in other vertebrate species. In mammals, for instance, these early events might be slightly different as the first visible sign of eye formation are two small depressions (optic pits) in the anterior neural tube, which appear well before folding of the tube occurs.

Independent of these initial differences, in all vertebrates the optic vesicles are formed by a single pseudostratified neuroepithelium that is surrounded by cephalic mesenchyme, including migrating neural crest cells, and limited at the most distal tip by the surface ectoderm. The cells that compose the optic vesicle neuroepithelium are initially morphologically and molecularly indistinguishable but reach their final complexity through a series of inductive and morphogenetic events that, in part, depend on their interaction with the surrounding tissues. The extension of thick cytoplasmic processes emanating from the basal surface of both optic vesicle and ectodermal cells establishes cell-to-cell contact and initiates a series of temporally correlated structural changes in both structures. A fibrillar extracellular matrix (ECM) builds up between the neuroectoderm and the ectoderm, favoring strong adhesion, while the ectoderm immediately adjacent to the optic vesicle thickens forming the lens placode (Figure 2).

Once formed, the lens placode and the adjacent optic vesicle begin a coordinated invagination to form the lens pit and the optic cup. ECM-mediated adhesion between the two tissues is important for coordinating morphogenetic movements. In addition, some of its components, such as laminin and fibronectin, may participate in lens cell differentiation, which occurs concomitantly. As a result, the lens pit deepens and gradually assumes a spherical shape that finally breaks away from the ectoderm forming the lens vesicle. A spatio-temporally restricted massive apoptosis contributes to this separation. Thereafter, the lens vesicle undergoes an asymmetric differentiation acquiring a characteristic polarity: cells located in the most proximal region (facing the neuroepithelium)

Figure 1 Optic vesicle morphogenesis in zebrafish. (a) The eye field (in green) in the anterior neural plate is surrounded anteriorly and peripherally by telencephalic precursors (blue), posteriorly by the dorsal diencephalon (light yellow) and medially by cells that will form the hypothalamus (red). (b, c) The hypothalamic and dorsal diencephalic precursors move forward beneath the eye field displacing it to the front and laterally. The lateral edges of the neural plate fold toward the midline. (c, d) The lateral telencephalon cells meet at the dorsal midline, leaving two optic vesicles at the side. Adapted from Egland, S. J., Blanchard, G. B., Mahadevan, L., and Adams, R. J. (2006). A dynamic fate map of the forebrain shows how vertebrate eyes form and explains two causes of cyclopia. Development 133: 4613–4617.

elongate to form the primary lens fibers, whereas those in the most distal portion (facing the ectoderm) form a cuboidal monolayer known as the anterior lens epithelium.

Concomitant with lens formation, the optic neuroepithelium undergoes a series of rearrangements that culminates with the formation of a bi-layered optic cup (Figure 2(c) and 2(c0 )). The distal inner layer will give rise to the future neural retina (NR), while the distal outer layer to the retinal pigmented epithelium (RPE), which acquires specialized epithelial characteristics. The proximal ventral region forms the optic stalk, from which the optic nerve derives. The transition zone between the future retina and the RPE forms the ciliary margin (CM), or periphery of the retina. Despite its neural origin, the CM differentiates into non-neural structures: the proximal part into the ciliary epithelium while the distal part becomes the iris. In fish and amphibians, the CM harbors bona fide stem cells, which continue to proliferate throughout life to increase the eye size. Cells with stem-like properties also appear to be present in the CM of birds and mammalian eyes. These cells, however, are quiescent in physiological conditions.

Although there are common features in the way the optic cup is formed among vertebrates, differences do exist. In fish and amphibians, the optic vesicles, which develop as flat wing-like protrusions, undergo a process of cavitation, rotation, and invagination that shifts cells that were originally in a ventral position to the medial layer, from which the RPE progenitors derive. These progenitors stretch over the prospective NR, occupying the dorsal aspect of the optic cup. In mammals and birds instead, the optic vesicle neuroepithelium undergoes two simultaneous foldings (Figure 2(b)): (1) the spherical vesicle becomes concave outward, resembling the bowl of a spoon where the optic stalk is the handle and (2) at the same time, the ventral portion of the entire vesicle folds inward giving rise to the groove of the optic (choroid) fissure. Differential growth rates are associated with these events. In the future RPE as well as in the CM, the proliferation rate is quite slow in comparison to that of the perspective NR. There are also differences along the dorso-ventral axis, with an initial higher proliferation rate in the ventral part, which enables the formation of the optic fissure.

The optic fissure can be divided into two adjoining parts – the retinal fissure and the optic groove – which derive from the progressive invagination of the ventral surface of the optic vesicle and stalk, respectively. The transition between the retinal fissure and the optic groove dictates the position where the optic disk, or blind spot of the retina, will form. This structure, a real interface between the optic stalk and the retina, enables the entrance of surrounding mesenchymal cells into the developing eye chamber, which will form the hyaloid artery, the main blood supply for the eye. At the same time, it allows the egression of retinal axons from the eye cup.

As a last step in the formation of a complete optic cup, its dorsal portion begins to proliferate more rapidly enabling the shallow bowl to become deeper, while its lateral edges meet ventrally across the optic fissure. The eye rudiment now has the appearance of a hemispheric cup where the RPE completely surrounds the NR. The sealing of the optic fissure completes these morphogenetic events (Figure 2(c) and 2(c0 )). Although this is a poorly understood process, in humans the fusion usually begins in the middle portion of the fissure and extends anteriorly and posteriorly.

Early Patterning

The morphogenetic movements and the tissue interactions described above are orchestrated by specific genetic programs coordinated by cell-signaling mechanisms – inductive signals – and regulated by the activity of a relatively small number of transcription factors (see Table 1). Many of the genes involved in the specification of the eye

Table 1 Summary of extracellular molecules and transcription factors implicated in early eye development

Shh, Sonic hedgehog; FGFs, Fibroblast grow factors; BMPs, bone morphogenetic proteins; RA, retinoic acid.

field, including retinal homeobox (Rx), paired box 6 (Pax6), hairy enhancer of split 1 (Hes1), orthodenticle homolog 2 (Otx2), LIM homeobox 2 (Lhx2), and SIX homeobox 3 (Six3), are also involved in the early patterning events that convert eye-field cells into the structures that compose the optic cup (Figure 3(a0 )). However, changes in the expression levels, as well as combinatorial interaction with other factors, diversify the activity of these genes enabling the progressive acquisition of different fates and functions. As mentioned above, cells that compose the optic vesicle are initially morphologically and molecularly undistinguishable and, therefore, are all potentially competent to originate the NR, the optic stalk, or RPE. Information, in the form of signaling molecules, derived from the surrounding tissues and the neuroepithelium itself modulates and restricts the expression of different transcription factors to a specific domain of the vesicle, initiating its patterning along the proximo-distal and dorso-ventral axis.

Proximo-Distal Patterning of the Optic Vesicle

The optic vesicles are bulges of the neural tube and thus their subdivision is often described according to a proximal–distal axis. In this view, future optic stalk precursors occupy a proximal location, while distal cells constitute the NR and RPE precursors (Figure 3(b)).

Proximo-ventral cells of the optic stalk are generated under the influence of signaling molecules, emanating from the axial ventral midline, including Sonic hedgehog (Shh) and nodal, a member of the transforming growth factor-beta (TGFb) family (Figure 3(b)). Mutations in these genes cause severe anterior neural tube defects that include the formation of a single midline (cyclopic) eye, lacking optic stalk tissue. The phenotype is the result of Shh-mediated regulation of the spatial expression of Pax6 and Pax2 – two homeobox transcription factors of the paired type – which normally

BMP/activin

D/V patterning of the optic vesicle

FGF

Induction of

RPE, NR, OS

Dorsal–distal

Otx2/1

Mitf

Pax6

Chx10

Rx Lhx2

Six3/6

Pax6

Pax2

Vax1

Ventral–proximal

D/V patterning of the neural retina

demarcate the distal and proximal optic primordium, respectively (Figure 3(b0 )). Indeed, overexpression of Shh causes the expansion of the Pax2 domain at the expense of the Pax6-positive tissue, while the opposite occurs in absence of Shh. A similar dependence on Shh signaling has also been observed for the expression of Vax genes, other homeobox transcription factors involved in optic stalk generation.

Targeted inactivation of Pax2 or Vax1 in mice results in severe optic nerve abnormalities, consistent with a role for these genes in proximal eye development. The phenotype of these mice is characterized by the presence of coloboma of the ventral optic fissure, and, in the case of Pax2, an extension of the RPE into the optic nerve region. In contraposition, the Pax6 gene is necessary for the

formation of the distal vesicle, including the lens tissue. Mutations in this gene impair both lens and optic vesicle formation, ultimately causing anophthalmia. In these mice, the only remnant of the eye primordium has optic stalk characteristics. Conditional inactivation of Pax6 at different time points of eye development, however, has highlighted additional functions for this gene both in NR and RPE development (see below).

In summary, acquisition of the ventral optic stalk phenotype depends on the Shh-dependent expression of transcription factors that impose ventral character to optic vesicle precursors. The establishment of a precise boundary between the presumptive optic stalk and the distal optic vesicle may result from the reciprocal transcriptional repression between Pax6 and Pax2 (Figure 3(b0 )).

Specification of the RPE and NR

Extracellular signals, derived from the surface ectoderm in contact with the prospective NR or from the periocular mesenchyme surrounding the presumptive RPE, pattern the distal optic vesicle (Figure 3(a)).

Removal of the surface ectoderm induces prospective NR cells to acquire the characteristics of RPE cells. This is because the ectoderm secretes high levels of two members of the fibroblast growth factor (FGF) family of signaling molecules – FGF1 and FGF2 – while the prospective retina expresses the FGF receptor 1. The addition of either one of the two factors is sufficient to rescue NR formation after ectoderm removal. On the contrary, exposure of the presumptive RPE to FGF confers to the cells NR properties. Therefore, FGF signaling normally activates NR specification but inhibits RPE formation. The future retina itself expresses other FGFs, such as FGF3, FGF8, FGF9, and FGF15, which may contribute to maintain the properties of the retina, but are not sufficient to induce them, since addition of these FGFs cannot rescue the phenotype caused by surface ectoderm removal.

The activity of the FGF is complemented by other signals, originating from the dorsal extraocular mesenchyme, which have opposite effects on the specification of these territories. Members of the TGFb superfamily of signaling molecules, such as activins or the related bone morphogenetic proteins (BMP) – Bmp4 and Bmp7 – are expressed in the surrounding mesenchyme and/or the presumptive RPE itself. Interference with the activity of these molecules prevents RPE development and induces NR-specific genes, whereas their addition activates RPE characteristics in the entire vesicle.

Thus, TGFb/BMP and FGF signaling act antagonistically on the specification of RPE and NR precursors. Directly or indirectly, their activity seems to impinge upon the expression of specific transcriptional regulators, such as Caenorhabditis elegans homeo domain 10 (Chx10) and Otx2 or microphthalmia-associated transcription factor (Mitf), the function of which is instrumental to the acquisition of either RPE or NR identities. Indeed, FGF signaling activates the expression of Chx10 in the retina but represses that of Otx2 and Mitf in the RPE, while opposite results have been observed with manipulations of TGFb/BMP signaling.

Besides those mentioned above, a number of additional transcription factors are also specifically expressed in the presumptive neural retina and RPE (Figure 3(b0 )). Currently, it is not clear how many of them are really needed to impose tissue specificity. The paired-like homeobox gene Chx10 (also known as Vsx1) is possibly the best candidate to impose a neural retina character to the naive optic vesicle cells (Figure 3(b0 )). The onset of Chx10 expression is restricted to the presumptive NR domain and mutations in the human or mouse gene

lead to microphthalmia, cataracts, and abnormal iris development. The expression of two members of the Six family of homeobox transcription factors, Six3 and Six6, initially expressed in the entire vesicle, is also soon restricted to the prospective NR. The overexpression of these factors in fish embryos causes retinal hyperplasia and ectopic formation of retinal-like tissue. In mice, genetic inactivation of Six3 leads to complete loss of the forebrain, while that of Six6 impairs retinal proliferation and differentiation. It is, therefore, still unclear whether the two genes are essential pieces of the genetic program that establishes NR identity. Nevertheless, overexpression of either one of them in the RPE cells leads to the acquisition of a NR phenotype, suggesting that their function is at least incompatible with the acquisition of the RPE character.

Similarly to Six3, the expression of Lhx2 becomes restricted to the prospective NR. Mice deficient in Lhx2 function are characterized by anophthalmia, like that observed in mice mutants for the Pax6 gene. Although the two genes seem to act independently, both are required during the period of close contact between the surface ectoderm and optic vesicle. Whether this implies that their activity participates in the specification of the NR domain still needs to be established.

The function of at least four transcription factors – Otx1, Otx2, Mitf, and Pax6 – is at the core of the transcriptional network required to establish the RPE character. Functional inactivation of either Mitf or Otx1 and Otx2 genes (bHLH and homeo-domain-containing transcription factors, respectively) impairs RPE differentiation. On the contrary, retinal cells transfected with either Otx or Mitf genes acquire a RPE phenotype and accumulate granules of pigment. Although it is unclear whether Otx and Mitf act in a feedback loop or in parallel, they synergize in the activation of melanogenic gene expression, including tyrosinase or the tyrosinase-related protein 2. Although Pax6 expression in the prospective RPE is transient, its function – possibly together with that of Pax2 – seems necessary to initiate Mitf expression. Indeed, Pax6 binds directly to the Mitf promoter and in embryos deficient in both Pax2 and Pax6 optic vesicles are small and composed of Mitfnegative cells that instead co-express Otx2 and the NR marker, Chx10.

As in the case of the proximo-distal patterning of the optic vesicle, subdivision of the distal vesicle in the NR and RPE is reinforced by reciprocal repression of transcription factors. Mitf is expressed ectopically in the neuroretina of Chx10-deficient mice, driving cells toward an RPE-like identity. Conversely, misexpression of Chx10 in the developing RPE caused downregulation of Mitf with a consequent loss of pigment, strongly suggesting an antagonistic interaction between Chx10 and Mitf.

Chx10 function also appears to be required to establish a boundary between central and peripheral retina, the CM. In Chx10 null mutants, in fact, peripheral structures, including the ciliary body, are abnormally expanded. Similar expansions are also observed upon stabilization of b-catenin, a key effector of canonical wingless pathway (Wnt) signaling. In addition, ectopic expression of Wnt ligand induces abnormal expression of CM markers, such as Otx1 and msh homeobox 1 (Msx1), in the central retina. This suggests that canonical Wnt signaling plays a crucial role in establishing a difference between central and peripheral retina, imposing identity to the ciliary body and iris.

Dorso-Ventral Patterning of the Optic Cup

As mentioned above, the transition between the optic vesicle and the optic cup establishes a new position for most of the optic vesicle precursors. Once this rearrangement is accomplished, the optic cup undergoes a new wave of polarization along the dorso-ventral axis.

A gradient of BMP signaling is primarily responsible for this new pattern (Figure 3(c) and (c0 )). Bmp4 is expressed in the dorsal retina of most vertebrate species analyzed. Mis-expression of Bmp4 in the ventral portion of the optic cup suppresses the expression of Pax2 and Vax and activates that of Tbx5, a transcription factor of the T-box class normally expressed only in the dorsal portion of the optic cup. The counterbalance for this dorsalizing activity is ventroptin, a BMP signaling antagonist expressed in the ventral optic cup. Forced expression of ventroptin in the dorsal cup decreases Bmp4 expression and expands that of Vax genes. Thus, the ventral character of the optic cup seems to depend on the inhibition of Bmp4-mediated dorsalizing activity.

Structures that are typical of the ventral optic cup – such as the optic fissure, the hyaloid artery, and the pecten (a vascular-like structure in birds) – also depend on BMP signaling, although this may be due to actions of other members of the family. Overexpression of the BMP antagonist noggin in the chick ventral optic cup alters its development and induces ectopic expression of optic stalk markers in the region of the ventral retina and RPE. Furthermore, in Bmp7 null mice the optic fissure does not form. Retinoic acid (RA) is an additional signal that may be involved in establishing the ventral optic cup identity, although its precise functions are controversial. Enzymes involved in the synthesis and degradation of RA as well as RA receptors are expressed in the eye with complex and polarized patterns. RA treatment upregulates Pax2 expression in the eye of zebrafish embryos, while deprivation of vitamin A during embryogenesis, inactivation of RA receptors, or inhibition of RA synthesis all lead to embryos with defects in the ventral retina. Despite this, embryos deficient

in Raldh1, Raldh2, and Raldh3, genes coding for RAsynthesis enzymes, develop an optic cup with a normal dorso-ventral patterning, questioning the relevance of RA in this process.

Equally unclear is whether Shh signaling contributes to impose a ventral character to the optic cup. Although Shh is needed to control the initial expression of Pax2 and Vax genes in the optic vesicle, its forced dorsal expression at slightly later stages of development does not ventralize the dorsal optic cup, although BMP4 expression is downregulated. Defects are, instead, observed in the optic disk and the NR, where Shh causes activation of Otx2 expression and pigmentation. Conversely, interference with Shh signaling perturbs Otx2 expression and pigment formation in the ventral RPE, which loses its characteristics. Thus, Shh signaling may have a more specific function in the specification of the ventral RPE cells as well as of those that compose the optic fissure.

Independent of the signaling mechanism, the transcriptional control of dorso-ventral polarization of the optic cup depends largely on the activity of Tbx5 in the dorsal portion of the cup and Vax genes in the ventral part. Overexpression of Tbx5 causes dorsalization of the optic cup while overexpression of Vax leads to its ventralization. Pax6 seems to regulate the expression of both genes but in opposite directions establishing an intermediate zone that separate the two domains.

The significance of a dorso-ventral polarity in the NR at this stage and, thus, of the function of Tbx5 and Vax genes might be tightly linked to the establishment of the proper spatial order of the retino-tectal projections through the regulation of the expression of members of the ephrin (Eph) family of receptor tyrosine kinases and their ephrin ligands, which are axon-guidance cues essential for target recognition of RGC axons.

Conclusions

Early eye patterning in vertebrates is controlled by an evolutionary conserved genetic program, although initial morphogenetic events may vary from species to species. This program is regulated by the interplay among a number of signaling pathways and transcription factors. The information available at present represents an important backbone that needs to be extended for full understanding of eye development. In vivo imaging of eye morphogenesis as well as additional characterization of the involved genetic networks may help to understand the causes of inborn eye malformation such as anophthalmia, microphthalmia, and coloboma.

See also: Developmental Anatomy of the Retinal and Choroidal Vasculature; Eye Field Transcription Factors;