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Charles Darwin appreciated the conceptual difficulty in accepting that an organ as wonderful as the vertebrate eye could have evolved through natural selection. He reasoned that if appropriate gradations could be found that were useful to the animal and were inherited, then the apparent difficulty would be overcome. Here, we review a wide range of findings that capture glimpses of the gradations that appear to have occurred during eye evolution, and provide a scenario for the unseen steps that have led to the emergence of the vertebrate eye.
To suppose that the eye, with all its inimitable contrivances… could have been formed by natural selection, seems, I freely confess, absurd in the highest possible degree… Yet reason tells me, that if numerous gradations from a perfect and complex eye to one very imperfect and simple, each grade being useful to its possessor, can be shown to exist… and if any variation or modification in the organ be ever useful to an animal under changing conditions of life, then the difficulty of believing that a perfect and complex eye could be formed by natural selection, though insuperable by our imagination, can hardly be considered real1.Charles Darwin (1809–1882)
More than 600 million years ago (Mya), early organisms evolved photoreceptors that were capable of signalling light, and that presumably mediated phototaxis, predator evasion by shadow detection or vertical migration, and the entrainment of circadian rhythms. However, it was not until the Cambrian explosion, beginning around 540 Mya, that animal body plans began evolving very rapidly2–4 and image-forming eyes and visual systems emerged. The possession of advantageous capabilities or attributes, such as sight, rapid movement and armour, might have become crucial to survival, and might have led to an ‘arms race’ in the development of defensive and offensive mechanisms5. In the various phyla eyes evolved with diverse forms, but apparently based on certain common underlying features of patterning and development, as exemplified by genes such as PAX6 and RAX (also known as RX), which have critical roles during neurulation and brain regionalization. For accounts of the origin and interrelationship of eyes in different phyla, see REFS 6–10.
Here we concentrate on the origin of the vertebrate eye. We summarize results from disparate fields: the emergence of the vertebrate (camera-like) eye, the evolution of photoreceptors, the phylogeny of opsins, the development of the eye cup, and the development of the retina. We then endeavour to integrate these findings to develop a plausible and coherent sequence for vertebrate eye evolution. To begin with, we summarize current ideas regarding the origin of vertebrates and the timescale involved.
FIGURE 1 illustrates a view of the evolutionary origin of vertebrates, in which the branching and timings are based primarily on morphological data and on the fossil record2,11–18. Although alternative patterns of branching and widely disparate timings have been derived from estimates based on ‘molecular clocks’ (that is, on the assumption that gene mutations have occurred at a fixed rate, or at least at a rate that can be estimated), we have instead based our datings on classical approaches because of the difficulties that have recently been demonstrated in the assumptions that underlie molecular-clock estimates18–21.
Primitive bilateral animals gave rise to protostomes (including most invertebrates) and deuterostomes (including our own line) approximately 580 Mya, although molecular-clock estimates suggest an even earlier separation. Subsequent progression in the deuterostome branch led to the emergence of chordates, which possessed a notochord (at least in the embryo) together with a dorsal nerve cord. Animals with a skull (craniates) emerged approximately 550–500 Mya. Of extant craniates, hagfish have the most basal body-plan. They possess neither jaws nor vertebrae and are therefore usually regarded not as vertebrates but rather as a sister group. The vertebrates comprise an early jawless (agnathan) division, of which the only living examples are lampreys, and a later jawed division, the gnathostomes, which includes fish and tetrapods.
Controversy has long surrounded the interrelationship between hagfish, lampreys and jawed vertebrates. BOX 1 summarizes current views, and in FIG. 1 we show hagfish diverging either before the divergence of lampreys or else after lampreys separated from the line that would become the jawed vertebrates.
Controversy has long surrounded the interrelationship between hagfish, lampreys and jawed vertebrates. One view is that hagfish are basal, having diverged before lampreys split from gnathostomes, whereas an alternative view is that hagfish have degenerated from a lamprey-like ancestor, with hagfish and lampreys forming an agnathan clade, the cyclostomes (round mouths) (FIG. 1). Here we list evidence and arguments that support each view, together with several unresolved issues.
On balance, and in the face of recent interpretations of the molecular evidence, we favour the view that hagfish are basal. In the words of Janvier141 (see also REF. 135): “All I can say is that, if cyclostomes are a clade, either hagfishes are the most extraordinary example of reversion among vertebrates, or lampreys and gnathostomes are the most extraordinary example of evolutionary convergence.”
Whatever the phylogenetic relationship between hagfish and lampreys turns out to be, it is very likely that improved knowledge of the eyes and photoreceptors of these species will help to refine our understanding of the origin of the vertebrate eye.
Not only has extensive gene duplication occurred throughout the evolution of animals22, but in addition it is widely accepted that two rounds of whole-genome duplication occurred early in vertebrate evolution23–29; most likely, one duplication occurred before the agnathans split from the vertebrate line and one occurred after (FIG. 1; for reviews, see REFS 30–32). It is also clear that the vertebrate organizer, which determines the body plan of developing embryos, arose in early chordates33–35. These genetic developments are likely to have been of crucial importance in early vertebrate evolution, but they are beyond the scope of this Review.
The eye of the adult lamprey is remarkably similar to our own, and it possesses numerous features (including the expression of opsin genes) that are very similar to those of the eyes of jawed vertebrates. The lamprey’s camera-like eye has a lens, an iris and extra-ocular muscles (five of them, unlike the eyes of jawed vertebrates, which have six), although it lacks intra-ocular muscles11. Its retina also has a structure very similar to that of the retinas of other vertebrates, with three nuclear layers comprised of the cell bodies of photoreceptors and bipolar, horizontal, amacrine and ganglion cells36,37. The southern hemisphere lamprey, Geotria australis, possesses five morphological classes of retinal photoreceptor and five classes of opsin, each of which is closely related to the opsins of jawed vertebrates38 (see below).
Given these similarities, we reach the inescapable conclusion that the last common ancestor of jawless and jawed vertebrates already possessed an eye that was comparable to that of extant lampreys and gnathostomes. Accordingly, a vertebrate camera-like eye must have been present by the time that lampreys and gnathostomes diverged (by the end of stage 4; see FIG. 1), around 500 Mya.
Might an even more ancient vertebrate-type eye remain in existence? Possibly so — in hagfish, which, as mentioned above, are a group of primitive, jawless, eel-shaped marine chordates.
Hagfish eyes39 are small, conical, completely lacking in any sign of a lens, an iris, a cornea or intra- or extraocular muscles, and buried beneath unpigmented translucent skin39,40. The retina contains only two main nuclear layers, with no obvious bipolar or amacrine cells, and the photoreceptors connect directly to the output neurons (ganglion cells)36,41–43. In hagfish, these ganglion cells project predominantly to the hypothalamus44,45, just as their likely homologues, the melanopsin-containing retinal ganglion cells, do in mammals46. Hagfish photoreceptors have a simple structure36,39,42,43 (FIG. 2), with rather poorly organized outer-segment membranes. In each of these respects the hagfish eye resembles the pineal organ of non-mammalian vertebrates47,48, with the exception that the hagfish eye is arranged bilaterally. Behaviourally, the hagfish seems to be almost blind, and its weak response to light is unaffected by removal of its eyes49. Thus, the hagfish ‘eye’ seems not to subserve vision; instead, it seems more likely to function as a circadian organ, similar to the gnathostome pineal complex (which hagfish lack).
The developmental changes that occur in the eyes of lampreys provide support for the notion that the ancestors of these animals succeeded those of hagfish in evolution. Lampreys have a larval form (the ammocoete) that develops slowly, over a period of 5 years or more, before metamorphosing into the adult50. The ammocoete is effectively blind, and its eyes are similar to those of the hagfish: they are small, they are buried beneath skin and they possess a relatively undifferentiated retina. Although the ammocoete retina contains a narrow central region (~50 μm wide, adjacent to the optic nerve) that is differentiated into the conventional three layers of vertebrate retinal neurons, the bulk of the larval retina remains undifferentiated, with a thick neuroblastic layer50–53. Over a period of years, a slow process of neural differentiation occurs, in the following sequence: ganglion cells, amacrine and horizontal cells, photo-receptors and finally bipolar cells51,52. This is broadly the same order as that in which the jawed-vertebrate retina differentiates54 (see below), although the ammocoete retina differentiates much more slowly. At metamorphosis, the lamprey eye grows considerably in size, the retina differentiates fully, the lens develops, the cornea splits into scleral and dermal layers 55 (allowing the eye to move with respect to the epidermis, as in jawed fish), extraocular muscles develop and the eye erupts at the surface to form a vertebrate-style visual organ.
This developmental progression is explained most parsimoniously if we assume that lampreys inherited their eyes from an ancestor that they had in common with hagfish, and that this hagfish-like larval eye is present in the larva but transforms to a vertebrate-like eye in the adult, and thus that lampreys arose from a hagfish-like ancestor. Alternatively, it is possible that extant hagfish correspond to an arrested form of lamprey development, and that hagfish are effectively a neotenous sister group to lampreys (BOX 1).
Although the hagfish eye is phylogenetically the earliest organ to exhibit a physical similarity to the vertebrate eye, clues to the molecular origin of the eye are retained in other extant organisms that diverged from the vertebrate line even earlier, such as amphioxus and Ciona intestinalis. Until recently it was thought that cephalochordates were the closest living relatives of vertebrates, with ascidia having diverged earlier; however, recent evidence has reversed this order, as illustrated in FIG. 1 (REF. 56). The tadpole-like larvae of sea-squirts (ascidians) such as C. intestinalis have a simple photosensory organ called an ocellus (FIG. 1). This ocellus contains a handful of ciliary photoreceptors (described below) surrounded by a single large pigment-containing cell57–59.
Many aspects of phototransduction and eye (or ocellus) development appear to be homologous between ascidians and vertebrates. Thus, the light-response of ascidian photoreceptors is hyperpolarizing60, as for vertebrates. Phototransduction in C. intestinalis uses a single opsin, Ci-Opsin1, which diverged from an ancestral ciliary opsin before the appearance of separate classes of vertebrate retinal and pineal opsins61, and a single class of arrestin that has close homology to cone arrestin62. In addition, the development of the ocellus requires the transcription factor Ci-Rx63, which is homologous to RAX, the highly-conserved transcription factor that is involved in vertebrate eye development63. Furthermore, the expression profile of transcription factors in the pigment cell of the ocellus resembles that in vertebrate retinal pigment epithelial (RPE) cells64. Finally, the ocellus contains homologues of three vertebrate proteins that are involved in the retinoid cycle of vision: RPE65, CRALB P and retinal G-protein-coupled receptor (RGR) opsin65. These and further observations strongly support the notion that a common ancestor of sea-squirts and vertebrates possessed a photoreceptive organ that contained many of the building blocks that are fundamental to light signalling in our own eyes.
The cephalochordate amphioxus has both ciliary and rhabdomeric photoreceptors, located in four separate non-imaging structures: the frontal ‘eye’ and lamellar body (ciliary), and the Joseph cells and dorsal ocelli (rhabdomeric)66,67. Amphioxus has six distinct opsins68, with the melanopsin-like opsin driving a Gq-based photo-transduction mechanism69, and the encephalopsin and peropsin homologues thought to drive Gt- and Go-coupled mechanisms, respectively68. like C. intestinalis, amphioxus has been the subject of extensive evolution–development studies that include the mapping of transcription factors70,71. One interesting possibility (see below) is that the rhabdomeric photoreceptors in an ancestral chordate similar to amphioxus might have received synaptic input from ciliary photoreceptors and subsequently evolved in craniates to become retinal projection neurons72.
The photoreceptor cells of animals can be classified into two types: rhabdomeric or ciliary. One of the main differences between these types relates to the topology of the large area of membrane in which the photopigment (opsin) is incorporated: rhabdomeric photoreceptors use apical microvilli, whereas ciliary photoreceptors extend membranes from a modified cilium (characterized by a 9+0 microtubule organization) (see schematic insets in FIG. 3). Rhabdomeric photoreceptors use a Gq- or Go-mediated G-protein-coupled signalling cascade that elicits a depolarizing response to light, whereas ciliary photoreceptors generally use a Gt-mediated G-protein cascade that elicits a hyperpolarizing response to light; however, in both classes of photoreceptor the graded voltage signals are transmitted across a synapse that is electrotonically close to the soma. The visual pigments of the two types of photoreceptor fall into two corresponding clades of rhabdomeric and ciliary opsins8,73, as discussed below.
Until recently, it was widely thought that rhabdomeric photoreceptors are present exclusively in protostomes (comprising most invertebrates), whereas ciliary photoreceptors are present exclusively in deuterostomes (including chordates). However, it is now clear that both types can coexist in a variety of organisms, including the chordate amphioxus67, the polychaete worm Platynereis72, the scallop74 and possibly insects75 — that is, in chordates, annelids, molluscs and arthropods, respectively (FIG. 1).
In analysing the phylogenetic relationships between opsins, and between the transcription factors that specify eye development, Arendt and colleagues concluded that the bilateral common ancestor of protostomes and deuterostomes (see stage 1 of FIG. 1) possessed both rhabdomeric and ciliary photoreceptors8,9,72. Furthermore, they proposed that vertebrate retinal ganglion cells are sister cells of rhabdomeric photoreceptors, on the basis of the close homology in the transcription factors used by the two classes of cell and the finding that melanopsin (which is contained in a sub-class of light-sensitive retinal ganglion cells) is a member of the rhabdomeric class of opsins9,72,76. Hence, it is entirely plausible to view our own retinal ganglion cells as rhabdomeric photo-receptors that have lost their rhabdomeric membrane structure, retained their axon, their ancestral responses to neurogenic factors and, in some cases, their rhabdomeric opsin and G-protein signalling cascade, and that have evolved the ability to receive synaptic input from ciliary photoreceptors.
The morphology of ciliary photoreceptors is illustrated in FIG. 2 at several ‘stages’ of chordate evolution, from tunicates to mammals. A smooth transition of features from stage to stage is apparent, compatible with the sequential acquisition of these features in ancestral forms. The stages we have illustrated are: the ascidian larva ocellus58,77, the hagfish eye36,39–43, the lamprey pineal organ48,78,79, the lamprey retina36,80–85 and the jawed-vertebrate retina.
In each case, the membrane that contains the photo-pigment extends from a cilium, and this region of the cell is denoted as the outer segment. In the ascidian larva (which reflects completion of stage 2), the membranes are organized approximately longitudinally with respect to the cell’s axis, rather like the petals in a rosebud. In hagfish (which reflect the end of stage 3), the membranes radiate more laterally, from a central cilium. In pineal photoreceptors (of lampreys), the cilium occupies an off-axis position and the outer-segment membranes appear a little more evenly stacked than in hagfish. In the retinal photoreceptors of vertebrates (jawless and jawed, reflecting completion of stage 4 onwards), the membranes are neatly stacked and highly ordered and the connecting cilium is lateral — that is, it is at the edge of the conical or cylindrical outer segment.
The ascidian photoreceptor (which arose by the end of stage 2) differs from the photoreceptors that arose at stage 3 onwards in other ways as well. For example, the neck of the ascidian photoreceptor (corresponding roughly to the inner segment) is surrounded by a pigment-containing cell, which appears to extend microvilli that interdigitate with the membranes of the outer segment. Thus, the pigmented cell in the ascidian larva has its soma in the same layer as the photoreceptors, rather than in a layer (such as the RPE) that has folded over the photoreceptor layer. Light normally reaches the ascidian photoreceptor from the outer-segment side, and the pigment granules shield the outer segment from light arriving from the opposite direction, so that the ocellus is of the everted type, rather than being inverted, as in the vertebrate eye.
In hagfish photoreceptors, the presynaptic terminal contains a synaptic density surrounded by synaptic vesicles36,43,86, whereas vertebrate photoreceptors are generally reported to exhibit a clearly-developed ribbon synapse. However, it has been reported that the vertebrate ‘ribbon synapse’ can in fact contain both spherical and ribbon synaptic bodies87,88. In both the hagfish retina and the vertebrate pineal organ, synaptic output occurs directly onto projection neurons (ganglion cells). In the retina of both jawless and jawed vertebrates the output occurs onto interneurons (horizontal and bipolar cells).
It has been known for many years that the retina of northern hemisphere lamprey species possesses two classes of retinal photoreceptor, that have either long (cone-like) or short (rod-like) outer segments80. On the other hand, the southern hemisphere lamprey, Geotria australis, has been shown to possess five morphological classes of photoreceptor, all of which are cone-like85, together with five classes of opsin38. This correspondence suggests that each class of photoreceptor might express a single class of opsin89, although RNA in situ hybridization will be needed to confirm this. Most non-mammalian vertebrates possess four classes of cone opsin, together with rhodopsin, which is expressed in rods, whereas mammals have lost two of these classes: SWS2 and Rh2. The recent results from G. australis38 indicate that at least four classes of opsin (long-wave sensitive (LWS), short-wave sensitive 1 (SWS1), SWS2 and an Rh opsin) evolved before the separation of lampreys and gnathostomes — that is, by the end of stage 4, around 500 Mya.
Together, these observations indicate a continuous transition in the characteristics of ciliary photoreceptors, over the time span represented by stages 1 to 6.
In studying the evolution of photoreceptors, it will ultimately be important to obtain a comprehensive understanding of the evolution of all of the components of the phototransduction signalling cascade, as well as of all of the factors that determine cell morphology. Although information about the parallel evolution of the many proteins that are involved in phototransduction is rapidly emerging90,91, the greatest progress to date has been made in relation to the opsin photopigments, on which we now concentrate.
FIGURE 3 shows a phylogenetic tree of important visual and non-visual opsins92. Before the separation of protostomes and deuterostomes, a primordial opsin had divided into three main branches: a rhabdomeric branch, a ‘photoisomerase’ branch and a ciliary branch. In vertebrates, the first two branches have given rise to melanopsin, which is expressed in retinal ganglion cells, and to RGR and peropsin, which are expressed in the RPE. The ciliary branch has given rise to many opsins, which are expressed in the pineal complex, in the retina and in deep-brain regions. The branching pattern of vertebrate retinal opsins that is shown in FIG. 3 was elucidated in 1992 by Shichida and colleagues, who deduced that all four classes of cone pigment were present before the evolution of the rod pigment, rhodopsin93.
Subsequent studies have shown how the roles of several key sites have altered during evolution94–97, and have defined the molecular characteristics of the different classes of opsin98–105. In almost all visual pigments the overall length of conjugated single and double bonds in the retinaldehyde chromophore is increased by the use of a protonated Schiff base bond between the chromophore and a lysine residue in the opsin. This bond must be stabilized by a nearby negative ion. In the presumed ancestral opsin, the counter-ion (Glu) was located at residue 181 (where it remains in rhabdomeric and photoisomerase opsins), but in the ciliary photoreceptors of C. intestinalis and craniates, the site of the counter-ion has moved to residue 113 (REF. 95). A negative ion is nevertheless retained at residue 181 in all ciliary opsins (except vertebrate ancient (VA) opsin), and there is evidence that this site is crucial to light activation and that the ion acts as the counter-ion in the photoactivated metarhodopsin-I state106.
Although the LWS opsins do not have Glu181 (FIG. 3), they nevertheless retain a negative charge by using a chloride-binding site, His181/Lys184 (REF. 107), as the secondary counter-ion. Although this change has had the benefit of extending the spectral sensitivity of LWS opsins further into the long-wavelength region, it has done so at the cost of greatly decreasing the thermal stability of the molecule.
Two amino acid sites, 122 and 189, have been identified as being especially important in distinguishing rhodopsin from cone opsins94,97. The rhodopsin of jawed vertebrates has a Glu at site 122 and an Ile at site 189, and site-directed mutagenesis has shown that these residues invest the molecule with a number of its rod-like properties: increased thermal stability, slowed decay of the photointermediates (the so-called meta-rhodopsins) and slowed regeneration of the pigment in darkness94,96,97.
Judging by the amino-acid residues at its site 122 and its site 189, lamprey ‘rhodopsin’ (RhA) is intermediate between rod and cone opsins96: it has the rod-type Glu122 but the cone-type Pro189 (FIG. 3). As mentioned previously, the photoreceptors that this opsin is incorporated into have cone-like morphology85; furthermore, both the short and long types of photoreceptor in the northern hemisphere lamprey, Lampetra fluviatilis, display properties that are cone-like: neither saturates at high light intensities80. In addition, it has recently been found that the phototransduction cascade in the photoreceptors of the southern hemisphere lamprey, G. australis, appears to be driven by a single set of cone-like phototransduction genes108. Together, these results suggest that the features of ‘true’ rod transduction in jawed vertebrates, which permit the reliable detection of single photons of light, evolved after the separation of gnathostomes from lampreys.
Early in the nineteenth century, Karl Ernst von Baer109 observed that organisms with widely different adult forms have embryos that closely resemble each other, and he suggested that the developmental stages through which the embryo passes might reflect the evolutionary history of the organism. Even though evolution selects from all stages of embryogenesis, the fundamental body plan that was established early in evolution does not often seem to be altered in a major way during embryogenesis; instead there appears to be a tendency for newly evolved features to be ‘tacked-on’ to later stages of embryogenesis. von Baer’s ideas were subsequently expanded upon by Ernst Haeckel110, to produce what is widely regarded as the extreme and simplistic view that ‘ontogeny recapitulates phylogeny’. Despite the controversy that was aroused by Haeckel, it nevertheless seems likely that a range of features in the development of the vertebrate eye reflect stages in its evolutionary history and, furthermore, that analysis of such apparently conserved features might permit the formulation of testable hypotheses about the morphogenetic mechanisms of the retina.
During vertebrate development the eyes arise as evaginations from the developing diencephalon (FIG. 4; animated online). Depressions called optic grooves form on each side of the rostral neural plate (FIG. 4b), and as the neural plate folds upwards and inwards (FIG. 4b,c), meeting and closing-over to form the neural tube, these growing regions balloon outwards (FIG. 4d) and are now termed optic vesicles. Contact between the expanding optic vesicle and the surface ectoderm (FIG. 4e), at a region termed the lens placode, induces changes in both tissues. The vesicle now invaginates to form the optic cup (FIG. 4f), while the ectoderm differentiates to form the lens pit and eventually the lens itself (FIG. 4g). Although it is sometimes suggested that invagination of the optic cup is a physical result of growth of the lens, this cannot be the case, because a normal eye cup and retina can develop in the absence of a lens, as in human congenital primary aphakia111. In our view, it seems plausible that a sequence broadly comparable to that described above might have occurred during the evolution of the vertebrate eye.
Recently a time-lapse imaging study of the developing zebrafish forebrain succeeded in tracking the cell migrations that occur very early in the development of the eye112. The animations that accompany this study provide impressive visualizations of the coordinated cellular migrations that are involved in eye formation.
One important aspect of the invagination of the optic cup is that it brings the future retina into contact with the future RPE (FIG. 4f). Thus, the pigmented RPE cells, which isomerize the chromophore retinal to its 11-cis form and synthesize melanin, which helps to attenuate unwanted light, now lie in the inverted position — ‘behind’ the photoreceptor cells (in terms of the path of incident light). Furthermore, they no longer occupy space in the photoreceptor layer, so that more of the light can be captured. These factors might help to explain the inverted nature of the vertebrate retina.
The folded arrangement of the vertebrate retina and RPE provides an evolutionary explanation for the occurrence of the choroid fissure, as proposed more than a century ago113. Early in evolution, before the optic cup invaginated, the axons from retinal ganglion cells would simply have run over the surface of the structure. Hence, one can view the optic nerve as having acted rather like a rope in linking the retina to higher centres: the developing eye cup has simply wrapped around this ‘rope’, and the developing axons have thereby not needed to penetrate the retina.
Finally, it is important to bear in mind that the other main photoreceptive regions of the vertebrate brain (the pineal and parapineal organs and the parietal eye) also arise as evaginations from the roof of the developing diencephalon. These organs are closely homologous with the lateral eyes, and a number of studies have highlighted the evolutionary significance of such parallels47,48,114–117.
With a degree of caution, one can interpret the developmental sequence described above, together with the findings set out earlier for ascidian, hagfish and lamprey eyes, as potentially providing clues about the evolution of the vertebrate eye. We now interpret these observations and propose the following scenario (see also BOX 2).
These protochordates had ciliary photoreceptors with a ciliary opsin and a hyperpolarizing response, and were able to regenerate 11-cis retinal in darkness.
The resulting paired lateral photoreceptive organs would have resembled the ‘eyes’ of extant hagfish, lacking any image-forming apparatus and subserving non-visual functions.
The resulting eye and visual system would have resembled that in extant lampreys and would have provided spatial vision at photopic intensities and over a broad wavelength range.
This eye possessed a duplex retina that contained both rods and cones, together with retinal wiring that closely resembled that of jawed vertebrates, with colour-coded photopic pathways and a dedicated scotopic pathway; it was probably similar to that found in many extant fish.
Before vision evolved, a common ancestor of hagfish, lampreys and jawed vertebrates possessed photosensitive ciliated epithelial cells that lined the ventricular surface of its brain. To aid light capture this region expanded, and in doing so part of it ballooned outwards laterally (see figure 7 of REF. 8). With the development of an overlying skull, the lateral light-sensitive region was better able to absorb light by expanding even further laterally. Light capture was additionally enhanced when an interaction between this ‘eye vesicle’ and the surface ectoderm, at what would become the lens placode, led to both invagination of the eye cup and the prevention of pigmentation of the overlying skin. At this stage of evolution (by the end of stage 3, around 530 Mya) these lateral light-sensitive regions corresponded broadly to the ‘eyes’ of extant hagfish, lacking any imaging apparatus and presumably having a circadian or shadow-detection function rather than any role in image-forming vision.
In an ancestor of vertebrates (but not of hagfish) further development of the lens placode occurred, leading to the formation of a functional lens. In addition, extraocular muscles evolved from both neural crest and mesodermal tissue. In extant lampreys, these developmental events are delayed until metamorphosis, whereas in extant jawed vertebrates they follow on seamlessly after the lateral expansion of the eyes. In addition to these developments in imaging capabilities, it seems that advances in signal processing also occurred. Clues to the possible origin of such advances might be found by examining the development of retinal circuitry.
Cells in the neuro-epithelium of the optic vesicle, which will generate the retina, proceed through the standard cycle of replication in much the same way as cells in other regions of the brain, with the cell body following a clearly defined pattern of migration during the cycle (FIG. 5a; for a review, see REF. 54).
During G1 interphase, the soma of the cell migrates towards the inner (vitread) surface (FIG. 5a). Here the cell undergoes S phase, synthesizing DNA and replicating its chromosomes. During the subsequent G2 interphase, the tetraploid nucleus migrates back towards the outer (ventricular or sclerad) surface. At this outer surface, the cell undergoes mitosis (M phase), separating into a pair of daughter cells, each of which can either exit the cell cycle (by entering the G0 phase) or continue through the cycle again. Once a cell has exited the cycle its soma migrates towards the final position that it will occupy in the retina once mature.
Three papers that were published in the late 1980s showed that dividing retinal progenitor cells can produce clones comprising any combination of cell types118–120. Thus, progenitor cells appear to be unrestricted in fate, with the capacity to form any class of retinal cell. There is currently intense research interest in the molecular mechanisms that underlie a retinal cell’s fate choice121–125.
The order in which different classes of retinal cell are ‘born’ (defined as the time at which they exit the cell cycle) is highly conserved across vertebrate species (FIG. 5b typifies the timing in mammalian retinae). During a first wave of cell genesis, retinal ganglion cells, horizontal cells, amacrine cells of at least one class and cone cells are born. A subsequent wave produces the remaining amacrine cells and also Müller cells. A third wave produces rod photoreceptors and finally bipolar cells (for a review, see REF. 54).
Initially (during the first post-natal week in the ferret) the photoreceptors have a simple bipolar form (FIG. 5c) and, in previous studies, they might have been incorrectly regarded as being uncommitted and/or undifferentiated. However, it is now clear that these young cells express markers that are specific for photoreceptors (for example, recoverin, vGluT1 and even rhodopsin) long before they exhibit the characteristic morphological features of mature photoreceptors. The somata of the young cones lie adjacent to the outer limiting membrane (OLM), whereas those of the young rods (which are born after the cones) lie slightly more vit-read. Each photoreceptor soma is linked to the OLM by a short process.
Importantly, each young photoreceptor also projects a process directly to the inner plexiform layer (IPL), passing without contact through the outer plexiform layer (OPL) that is just beginning to form, even though horizontal cells, with which the photoreceptors will eventually connect, have already adopted their final positions in the vicinity of the future OPL. These events are independent of the formation of Müller cells. Within the IPL the photoreceptor processes terminate in two discrete sub-layers, coincident with the stratifying processes of cholinergic amacrine cells. The photoreceptor terminals express the synaptic proteins synaptophysin and synaptotagmin, and have the morphological appearance of functional synapses126. However, it has not been established whether the post-synaptic processes that they contact are amacrine or ganglion cells.
Subsequent to the formation of transient contacts, at approximately post-natal day 14 in the ferret, these transient processes retract from the IPL and the photoreceptors instead make synaptic contacts in the developing OPL (FIG. 5d). This phase of retraction coincides with maturation of the bipolar cells and with continued maturation of horizontal cells and the OPL. At the developing OPL each photoreceptor terminal first forms a dyad contact with a pair of horizontal cell processes and is thereafter contacted by a bipolar cell process, giving rise to the adult triad arrangement of the ON pathway.
This developmental sequence, in which an initial direct synaptic contact from the photoreceptor to the ganglion cell is followed by the subsequent insertion of a bipolar cell between the two cells, suggests that a comparable sequence of events might have occurred during the evolution of the retina in the transition from a hagfish-like retina to a vertebrate-like retina109.
At approximately the time that synaptic contacts form at the OPL, the distal process of each photoreceptor penetrates the outer limiting membrane and begins to form an inner segment and an outer segment (FIG. 5e). Hence, it is not until at least this stage of retinal development that conventional visual transduction becomes possible.
Retinal bipolar cells share a large repertoire of features with rod and cone photoreceptors. Structurally, retinal bipolar cells have the same ‘bipolar’ shape that developing photoreceptors have before their inner and outer segments differentiate (FIG. 5c). Bipolar cells develop an appendage (the Landolt club) that bears a striking similarity to the outer segment of the photoreceptor: it has microtubules that are organized in the 9+0 pattern that is characteristic of non-motile cilia and it is located at the outer limiting membrane, although it lacks disc membranes. In terms of transduction mechanisms, the G-protein signalling cascade of the ON bipolar cells bears a remarkable similarity to the phototransduction cascade in photoreceptors. In addition, the output synapse of the bipolar cell is very similar to that of the photoreceptor, in that it uses synaptic-ribbon structures that are not found in other types of retinal neuron. Finally, many of the proteins that are expressed in bipolar cells are either identical to, or else represent isoforms of, proteins that are found in cone and rod photoreceptors — for example, recoverin, potassium channels and the molecular machinery of ribbon synapses.
One possibility that might account for these observations is that photoreceptors and bipolar cells are independent descendants of the ‘protoneuron’ that is proposed to have been the most ancient type of vertebrate nerve cell114,115. An extant representative of this protoneuron appears to be the sensory cerebrospinal fluid (CSF)-contacting neuron that is present in the CNS of lancelets, hagfish128 and lampreys129 as well as jawed vertebrates. An alternative scenario could be that bipolar cells evolved from ciliary photoreceptors. In this case one might anticipate that, whereas cone ON bipolar cells would have descended from cones, rod bipolar cells might have descended from rods, rather than from cone bipolar cells.
In addition to the transient embryonic contacts of photoreceptors onto ganglion cells described above, direct contacts from photoreceptors to ganglion cells are maintained in the adult retina. Biplexiform ganglion cells are a class of retinal projection neuron that seem to exist in all vertebrate species from lamprey to primate37,130–133, and which receive synaptic input from photoreceptors as well as from bipolar cells. In addition, the lamprey has a class of ganglion cell that might contact only photoreceptors132. Hence, it seems possible that such cells might represent a primordial form of retinal projection neuron that originated before the evolution of bipolar cells. The lamprey retina is also interesting in that the majority of ganglion cells (and nerve fibres) lie distal to, rather than proximal to, the IPL (see figure 7 of REF. 37). This might represent the primordial layering arrangement of the vertebrate retina, in which case an inversion of layers through altered patterns of radial migration might have occurred in the gnathostome retina.
Some of the most fundamental events in the evolution of the vertebrate eye occurred in the relatively short time of a few tens of millions of years, around the time of the Cambrian explosion more than 500 Mya and possibly coincident with two duplications of the entire genome.
First, it seems likely that a basal chordate possessed simple paired photoreceptive organs that were broadly similar to the unpaired organs of extant Amphioxus and C. intestinalis (it seems very likely that during evolution C. intestinalis has lost one member of what was once a pair77). by approximately 530 Mya these paired organs had expanded laterally, and each had developed into a two-layered ‘retina’, with ciliary photoreceptors contacting projection neurons (which might have arisen from rhabdomeric photoreceptor cells). The resultant organ would have been quite similar to the ‘eye’ of extant hagfish, with a circadian and/or shadow-detecting function and a lack of optical components for imaging. The advantage it conferred to the animal was the ability to gather much more light (possibly at great depth), through a large increase in the number of photoreceptors, through lateral positioning outside the skull and through de-pigmentation of the overlying skin.
Subsequently, in evolutionary steps that closely parallelled the developmental steps that occur in extant metamorphosing lampreys, this rudimentary eye acquired a lens, an increase in retinal processing power (through the insertion of retinal bipolar cells), projection of the ganglion cell axons to thalamic regions, and extraocular muscles. This eye, which was equivalent to the eye of adult extant lampreys, possessed almost all of the crucial features that characterize the modern vertebrate eye, and was present at least 500 Mya. Along with this transformation of the hagfish-like lateral organs from a non-imaging function to an imaging function, another region of the diencephalon expanded to take over circadian function and evolved into the pineal organ.
The evolutionary steps that we propose are set out as an explicit scenario in BOX 2, and it can be seen that a number of these steps are retained during the development of modern embryos. In our view a case can be made that a great number of these proposed steps are likely to have been advantageous to the animals that evolved them; indeed, for the optical features, an assessment of the performance of the various stages of eye cup and lens development has previously been presented134. Accordingly, we think that this proposed sequence might satisfy Darwin’s prescription for the evolution of the vertebrate eye.
We have conducted a comparative analysis of the features of opsins, photoreceptors, retinal connectivity and eye morphology, across organisms that range from sea-squirts to hagfish, lampreys and jawed vertebrates. By combining this analysis with a consideration of the embryological development of the mammalian eye, we have been able to discern what we believe to be a long sequence of transitions that we postulate have underlain the evolution of the vertebrate eye.
Our hypotheses are open to test, because they lead to a number of explicit predictions, several of which we present in BOX 3. Clearly, a great deal rests on establishing the precise status of the hagfish: that is, on clarifying the details of its retinal circuitry and its correct phylogenetic position. By undertaking experiments of the kind indicated in BOX 3, it should be possible, within a relatively short time, to support, revise, or rule out the ideas that we have gathered here.
Prediction 1: the phototransduction cascade components of tunicate ocelli should be homologous with those of hagfish and lamprey photoreceptors.
Prediction 2: at an early stage of eye evolution there was synaptic contact from ciliary photoreceptors onto rhabdomeric photoreceptors.
Prediction 3: hagfish photoreceptors should exhibit close homology to cones.
Prediction 4: the hagfish retina should not contain bipolar cells, and its photoreceptors should synapse directly onto the projection neurons (ganglion cells).
Prediction 5: if hagfish are monophyletic with lampreys, then they might represent a form with arrested development, rather than a degenerate form.
Prediction 6: lampreys ought not to possess true rods.
Prediction 7: lampreys should not possess rod bipolar cells.
Prediction 8: if vertebrate bipolar cells are descended from photoreceptors they will share numerous molecular components or have very close homologues.
We wish to thank T. Cronin, I. Meinertzhagen, S. Conway Morris and P. Janvier for constructive comments on the manuscript. This work was supported by the Australian Research Council (FF0344672; T.D.L. and S.P.C.) and by the US National Institutes of Health and the Research to Prevent Blindness Foundation (E.N.P.).
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Trevor D. Lamb's homepage: http://jcsmr.anu.edu.au/org/dns/visual/index.php
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