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

Histogenesis: Cell Fate: Signaling Factors; Photoreceptor Development: Early Steps/Fate; Retinal Histogenesis; Zebra Fish–Retinal Development and Regeneration.

Further Reading

Adler, R. and Canto-Soler, M. V. (2007). Molecular mechanisms of optic vesicle development: Complexities, ambiguities and controversies.

Developmental Biology 305: 1–13.

Barishak, Y. R. (2001). Embryology of the Eye and Its Adnexa. Basel: Karger.

Chow, R. L. and Lang, R. A. (2001). Early eye development in vertebrates. Annual Review of Cell and Developmental Biology

17: 255–296.

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.

Fitzpatrick, D. R. and van Heyningen, V. (2005). Developmental eye disorders. Current Opinion in Genetics and Development 15: 348–353.

Lupo, G., Harris, W. A., and Lewis, K. E. (2006). Mechanisms of ventral patterning in the vertebrate nervous system. Nature Reviews Neuroscience 7: 103–114.

Martinez-Morales, J. R., Rodrigo, I., and Bovolenta, P. (2004). Eye development: A view from the retina pigmented epithelium. BioEssays 26: 766–777.

Rembold, M., Loosli, F., Adams, R. J., and Wittbrodt, J. (2006). Individual cell migration serves as the driving force for optic vesicle evagination. Science 313: 1130–1134.

Relevant Website

http://www.med.unc.edu – University of North Carolina at Chapel Hill, School of Medicine: Eye Development.

Glossary

Bilaterian – A major family of animals characterized by bilateral symmetry, including insects, nematode worms, mollusks, vertebrates, echinoderms, and other animals.

Chromophore – A light-reactive chemical that binds to a protein, such as opsin, and which allows biological sensitivity to light.

Ciliary – Relating to cilia, which are subdivided into motile and sensory types. Ciliary photoreceptors are those in which phototransduction occurs in a sensory cilium.

CNG (cyclic nucleotide-gated ion channel) –

A family of proteins that acts (along with other functions) as the ion channel in ciliary photoreceptor cells. CNG proteins open in the presence of cyclic nucleotides to allow positively charged ions such as calcium to flow into the cell.

Cnidaria – A group of animals that includes jellyfish, anemones, hydras, and corals.

Co-duplication – The simultaneous duplication during evolution of interacting components (e.g., genes or proteins).

Co-option – The pattern or process whereby existing components (e.g., genes or proteins) are used in the evolution of a new structure or function. Eumetazoa – A major group of animals characterized by the presence of numerous cell types, tissues, and organs and that includes bilaterians and cnidarians.

GPCRs – Guanine-nucleotide-binding protein (G-protein)-coupled receptors form a class of proteins that functions by embedding in cell membranes. These proteins function to detect a signal outside the cell and then signal to proteins inside through a G protein to the cell to change the cell’s state in some way.

G protein (Gt, Gq, Go, Gs) – A family of multi-unit proteins that are activated by GPCR receptor proteins to transmit signals into cells. Gt, Gq, Go, and Gs are subfamilies of G-alpha proteins activated by opsins.

Photopic vision – The vision in high light levels, which may include color vision. Phototransduction – The conversion of a light signal received by an animal opsin to an electrical signal that is transmitted to the nervous system.

Rhabdomeric – A type of photoreceptor cell with numerous microvilli. It is also called microvillar photoreceptors.

Rhodopsin – A name originally given to isolated visual pigments that contained both opsin protein and nonprotein chromophore. Today the term still applies to visual pigments, and is also used commonly to describe the opsin protein expressed in vertebrate rod (dim-light) photoreceptors, and the opsins of certain organismal groups, such as bacteria.

Scotopic vision – A monochromatic vision in low light levels.

Spectral tuning – The changes in specific amino acids of opsin proteins that mediate the maximum sensitivity to different wavelengths of light of different visual pigments.

Tetrachromatic – A visual system based on four classes of visual pigments with four different l-max values.

Trichromatic – A visual system based on three classes of visual pigments with three different l-max values.

TRP (transient receptor potential ion channel) –

A family of proteins that acts (along with other functions) as the ion channel in rhabdomeric photoreceptor cells. TRP proteins open in the presence of products derived from the lipid diacylglycerol to allow positively charged ions to flow into the cell.

Type I opsin – The retinylidene proteins present in bacteria and algae, which are referred to by various names, including bacteriorhodopsin, bacterial sensory rhodopsins, channelrhodopsin, halorhodopsin, and proteorhodopsin. These proteins have varied functions, including bacterial photosynthesis

(bacteriorhodopsin), which is mediated by pumping protons into the cell, and phototaxis (channelrhodopsin), which is mediated by depolarizing the cell membrane.

Type II opsin – The retinylidene proteins present for approximately 600 million years in eumetazoans (animals not including sponges), and unknown from sponges or any nonanimals. The proteins of this family have varied functions, including phototransduction and vision, circadian rhythm entrainment, mediating papillary light reflex

(pupil constriction), and photoisomerization (recycling the chromophore).

l-max – The wavelength of light to which a visual pigment (opsin plus chromophore) is maximally sensitive.

Introduction

Opsins are a group of proteins that underlie the molecular basis of various light-sensing systems, including phototaxis, circadian (daily) rhythms, eye sight, and a type of photosynthesis. They are sometimes called retinylidene proteins because they bind to a light-activated, nonprotein chromophore called retinal (retinaldehyde). Opsins are also, in some cases, called rhodopsins, a name originally given to isolated visual pigments that contained both opsin protein and nonprotein chromophore in a time before the two separate components were known. Today, the term rhodopsin is used commonly to describe the opsin expressed in vertebrate rod (dim-light) photoreceptors, and the opsins of certain organismal groups, such as bacteria.

General Opsin Structure and Function

Opsins are light-sensitive proteins that snake in and out of a cell membrane 7 times. Crystal structures of opsins have been generated, which allows a detailed understanding of this protein structure. Opsin light sensitivity is mediated by light-sensitive chemicals called chromophores. Opsin proteins covalently bind a chromophore through a Schiff base linkage to a lysine amino acid in the seventh membranespanning region of the protein. The absorption of a photon of light results in a photoisomerization, or shape change, of the chromophore. Photoisomerization then causes a shape change in the liked opsin protein.

Type I and Type II Opsins

Two major classes of opsins are defined and differentiated based on primary protein sequence, chromophore chemistry, and signal transduction mechanisms. Several lines of evidence indicate that the two opsin classes evolved separately, illustrating an amazing case of convergent evolution.

Type I opsins are present in bacteria and algae and are referred to by various names, including bacteriorhodopsin, bacterial sensory rhodopsins, channelrhodopsin, halorhodopsin, and proteorhodopsin. These opsins have varied function, including bacterial photosynthesis (bacteriorhodopsin),

which is mediated by pumping protons into the cell, and phototaxis (channelrhodopsin), which is mediated by depolarizing the cell membrane. Type II opsins are present in eumetazoans (animals not including sponges), but are unknown from sponges or any nonanimals. As opsins are known from cnidarians and bilaterian animals (animals with bilateral symmetry, including humans, flies, and earthworms), type II opsins are inferred to have been present in their common ancestor, which lived about 600 million years ago. These opsins have varied function, including phototransduction and vision, circadian rhythm entrainment, mediating pupillary light reflex (pupil constriction), and photoisomerization (recycling the chromophore).

Despite their functional similarity and despite both being seven-transmembrane proteins, multiple lines of evidence indicate that type I and type II opsins evolved independently. First, the primary amino acid sequences of type I and type II opsins are no more similar than expected by chance. Second, the orientation of the transmembrane domains differs between the major groups. Third, the major opsin groups differ in chromophore chemistry. Prior to light activation, the chromophore of type I opsins is an all-trans isomer. Light activation then involves isomerization of the chromophore to 13-cis retinal. In contrast, prior to light activation, the chromophore of type II opsins is 11-cis retinal. Light activation of type II opsins involves isomerization to all-trans retinal (Figure 1). Fourth, type II opsins belong to the larger protein family called G-protein- coupled receptors (GPCRs), which transmit varied signals from outside to inside cells by activating guanosine triphosphate (GTP)ase proteins, which in turn signal to second messengers that affect the state of the cell in various ways. Type I opsins do not activate G proteins. Furthermore, type II opsins are more closely related to nonopsin, lightinsenstive GPCRs than they are to type I opsins.

Major Type II Opsin Classes

Based on phylogenetic analyses, there are four major classes of type II opsins in animals: cnidops, retinal G-protein receptor (RGR)/Go, rhabdomeric (Gq), and ciliary (Gt) (Figure 2).

Cnidops

In 2007, a new major class of opsins was described based on analyses of whole genome sequences of the phylum Cnidaria, which includes sea anemones and jellyfish. This cnidarian-specific class of opsins is called cnidops. The molecular details of cnidops-mediated phototransduction are not yet fully elucidated, but box jellyfish cnidops appears to activate Gs-class G proteins, which in turn activate adenylyl cyclase. For what might cnidarians be using opsins? Some jellyfish are known to use light cues to

Type II opsins

Octopus retinochrome

Human melanopsin

Rhabdomeric opsins (Gq)

Octopus visual opsin

regulate their depth in the water column and their swimming speed, while others (box jellyfish) possess sophisticated camera eyes analogous those of humans. These complex cnidarian eyes may be capable of image-forming vision, and are used for obstacle avoidance and perhaps for hunting prey. At present, there is no evidence for cnidops-mediated color vision.

Retinal G-Protein Receptor/Go

Retinal G-protein receptor (RGR) and Go opsins are functionally distinct from each other, but sometimes group together in phylogenetic analyses, indicating they may share more recent common ancestry with each other than with the other major opsin classes. Opsins grouping in this clade include those from vertebrates, cephalochordates (invertebrate chordates represented by the lancelet Branchiostoma), and mollusks.

In humans, three opsins often fall within the RGR/Go group: peropsin, neuropsin, and RGR. Cephalopod mollusks also possess a related opsin called retinochrome. Interestingly, members of this opsin class have not been noted from any arthropod or any other ecdysozoan (a group of molting invertebrates, including arthropods and nematode worms).

The RGR opsins are not well understood, but some important clues to their function exist. These opsins preferentially bind an all-trans isomer of retinal, contrasting with rod and cone opsin, which require 11-cis retinal for function. When tested in specific wavelengths of light that ranged from blue to ultraviolet (UV), RGR was able to convert all-trans retinal to the 11-cis isomer. Thus, it would seem that RGRs function to provide fresh retinal of the functional 11-cis conformation that is required for proper rod and cone opsin function. Retinochrome opsins from mollusks also have this photoisomerase function.

In addition, in mollusks and cephalochordates are opsins that may interact with a Go-class G protein, and as such are called Go opsins.

Rhabdomeric (Gq)

Opsins belonging to the rhabdomeric (Gq) class are present in bilaterian animals (animals with left and right sides), but unknown from Cnidaria. Where known, these opsins activate Gq-class G proteins, which drive phospholipase C second messengers. The opsins are sometimes called rhabdomeric opsins, because they tend to be expressed in specific cell types (rhabdomeric photoreceptor cells) that increase cell surface area using microvilli. Gq opsins are well known for mediating vision of many invertebrate animals, and are particularly well studied in the compound eyes of the fly Drosophila melanogaster. In chordates, including humans, Gq opsins are not expressed in the primary visual photoreceptors; instead, a Gq opsin,

called melanopsin is known to be expressed in retinal ganglion cells, which are among the cells that relay signals from the primary photoreceptors to the optic nerve. Melanopsin is also involved in mediating the papillary reflex.

Ciliary (Gt)

Opsins belonging to the ciliary (Gt) class are present in eumetazoan animals, including vertebrates, annelid worms, and cnidarians (anemones and jellyfish). In vertebrates, these opsins activate Gt-class G proteins (transducins), which drive phosphodiesterase (PDE) enzymes. In invertebrates, the G protein that is activated by ciliary opsins is unknown. This class of opsins is often called ciliary opsins because they are known to be expressed in ciliary photoreceptor cells, which assemble a phototransduction organelle from a sensory cilium membrane. In annelids and bees, ciliary opsins are expressed in the brain. Although the organismal function of ciliary opsins is unknown in invertebrates, some have suggested a role in entraining circadian rhythms. In vertebrates, ciliary (Gt) opsins are expressed in the primary photoreceptors, the rods and cones, with different subclasses of opsin expressed in rods versus cones and further subclasses expressed in different cones to mediate color vision. Ciliary opsins with extraretinal expression are also known from vertebrates, and have been given various monikers, often named for their site of expression, such as pinopsin, parapinopsin, and tmt opsin. Details about the molecular and organismal functions of these extraretinal ciliary opsins are scarce (Table 1). One opsin in this class, parietopsin, is expressed in the parietal eye of a lizard and activates a Go-class G protein.

Opsin and Color Vision

Color vision, the ability to discriminate between light information of different wavelengths, is present in various groups of animals including insects and vertebrates, and involves opsin genes. Although color vision is a behavioral phenomenon that involves processes of both physical detection and neurobiological comparison, duplicated opsin proteins expressed in the retina directly mediate the physical detection of different wavelengths of light.

The specificity of a given opsin protein to a given wavelength of light is summarized by its l-max, a measure of the peak absorbance across the light spectrum. The specific l-max of a given opsin protein is largely a function of its amino acid sequence. Opsins representing the different spectral classes are specifically expressed in individual photoreceptor neurons called cone cells. When stimulated by light of the proper wavelength, opsins trigger a cellular response, leading to a change in the resting electric charge, or potential, of a photoreceptor cell. Color vision emerges from this system through the comparison

of the resultant potentials of cone cells with those that express opsins of differing l-max expressed in adjacent cone cells. For instance, humans possess three cone-opsins with l-max values falling roughly into the categories of blue, blue–green, and yellow–green. Compared to other vertebrates, human opsins represent a single blue S opsin and two M opsins (blue–green and yellow–green) that resulted from a primate-specific gene duplication event. When red light falls upon a cone cell that is sensitive to yellow–green, it activates opsin signaling because light of this wavelength falls within the activity spectrum of this protein. However, red light does not fall within the sensitivity spectra of either the blue or blue–green opsins. Together, comparisons made between activated and nonactivated cone cells lead to the human perception of red light. In most vertebrates, these comparisons are made by additional cells in the retina (i.e., the horizontal and ganglion cells) and also in a portion of the brain called the visual cortex.

In vertebrates, four distinct opsin classes, each with distinct sensitivities, accomplish the full spectral range. These include violet-(V) short-(S), middle-(M), and long-

(L) wavelength varieties. Studies in the sea lamprey, a distant vertebrate relative of humans, have provided data that allow scientists to infer that the four major classes of vertebrate retinal opsins are the result of ancient gene duplication mutations that likely occurred before the

origin of all living vertebrates. This implies that major opsin clades must have been lost in various vertebrate lineages. For instance, placental mammals likely originated from nocturnal ancestors that lost the V and M spectral classes of opsin. In addition, some marine mammals are known to have further lost the S class of opsin and are cone monochromats. Similar losses have been reported in a deep-dwelling, Lake Baikal fish. In general, each of these examples of opsin loss can be correlated to photoecology. When spectral sensitivity of a given wavelength is not required, selection against mutations at these loci is relaxed, allowing the loss of functional copies. However, on numerous occasions, photoecology has provided a generative context for the origination of new opsin genes and classes in vertebrate and invertebrate lineages alike. Often, these new visual repertoires converge both in spectral sensitivity (i.e., l-max) and in the specific mutations that have lead to the functional shift.

Despite major differences between insect-faceted eyes and vertebrate camera-like eyes, both eye types utilize opsins in a similar way to achieve color vision. Similar to vertebrate color vision, insect color vision is based on a process of physical detection of light by opsins of differing l-max, followed by neurobiological comparison of these light data. In insects and other arthropods, the neurobiological basis for color vision is less well understood but

likely involves a part of the insect brain called the medulla. While the neurobiological basis for insect color vision requires further clarification, it is clear that opsin gene duplication has been a driving force for the evolution of color vision in insects and other invertebrates. For example, the duplicated opsin genes of fruit flies are well studied. Two fly opsins are maximally sensitive to UV (345, 375 nm), one to blue (437 nm), two to blue–green (420, 480 nm), and one to green (508 nm) light.

Molecular Basis of Wavelength Sensitivity

Different l-max values of different opsins are mediated by specific amino acid differences, which in some cases have been experimentally demonstrated. For example, in primates, three amino acid sites have a dominant role in differentiating red-sensitive and green-sensitive opsins. In birds, one opsin gene is blue sensitive in some species, and UV-sensitive in other species, a difference that is mediated by a single amino acid change. Changing the 84th amino acid of a zebra finch opsin allowed the protein’s sensitivity to change from UV to blue when the protein was expressed in cultured cells. Conversely, pigeon and chicken blue-sensitive opsins became UV sensitive in cell culture after a change of the 84th amino acid. The combination of gene duplication and differential changes in amino acids of duplicated opsins has generated a diversity of proteins in different species.

Opsin and Modes of Phototransduction

Evolution

As discussed above, opsin genes were very often duplicated and retained during animal evolution. Early opsin gene duplications led to the major opsin groups and more recent duplications mostly led to additional specializations, such as the ability for color vision. As members of highly coordinated protein networks, changes in opsin proteins are sometimes correlated with changes in partnering proteins. The interaction of two evolutionary processes has resulted in the diversity of opsin-based phototransduction pathways observed today that contains a combination of shared and distinct interactions. First, co-option refers to instances where an opsin recruited different intracellular signaling components than its ancestor during evolution. Second, coduplication involved the simultaneous duplication of multiple genes of an ancestral network. Co-option and coduplication are not discrete alternatives; instead, some genes of a network originated by co-duplication, whereas others joined the network by co-option. This becomes especially clear if we examine the evolution of particular networks at multiple timescales, or at increasing spatial scales by increasing the number of interactions considered.

An example of phototransduction pathways that evolved largely by co-duplication is the different pathways used in rod and cone cells of vertebrate retinas. Rods are specialized for dim light (scotopic) vision, and cones are specialized for bright light (photopic) vision. Rods and cones utilize different, duplicated opsins as well as different duplicated genes downstream of opsin. Multiple rodspecific and cone-specific genes originated through largescale segmental duplication of an ancestral vertebrate genome before the origin of gnathostomes (jawed vertebrates) and after the split of vertebrates and their closest nonvertebrate relatives. As such, the duplication of multiple genes in these pathways maps to the same time interval and is consistent with co-duplication.

Examples of the origin of new opsin pathways involving co-option probably include the origin the major opsin groups, which signal to different G proteins. In particular, the rhabdomeric opsins are the only opsins to signal through Gq G proteins and transient receptor potential (TRP) ion channels. Since rhabdomeric opsins originated in the ancestor of living bilaterally symmetric animals, and are unknown from Cnidaria, they have a more recent evolutionary origin than ciliary opsins, which are present in Cnidaria. This pattern indicates co-option: at or near their origin, rhabdomeric opsins began signaling to Gq and TRP, proteins that predate bilaterian animals.

See also: Circadian Rhythms in the Fly’s Visual System; Color Blindness: Inherited; The Colorful Visual World of Butterflies; Genetic Dissection of Invertebrate Phototransduction; Limulus Eyes and Their Circadian Regulation; Microvillar and Ciliary Photoreceptors in Molluskan Eyes; The Photoreceptor Outer Segment as a Sensory Cilium; Phototransduction in Limulus Photoreceptors; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Polarized-Light Vision in Land and Aquatic Animals; Rod and Cone Photoreceptor Cells: Inner and Outer Segments.

Further Reading

Arendt, D. (2003). Evolution of eyes and photoreceptor cell types.

International Journal of Developmental Biology 47(7–8): 563–571. Briscoe, A. D. and Chittka, L. (2001). The evolution of color vision in

insects. Annual Review of Entomology 46: 471–510.

Fernald, R. D. (2006). Casting a genetic light on the evolution of eyes. Science 313(5795): 1914–1918.

Hardie, R. C. and Raghu, P. (2001). Visual transduction in Drosophila. Nature 413(6852): 186–193.

Lamb, T. D., Collin, S. P., and Pugh, E. N. (2007). Evolution of the vertebrate eye: Opsins, photoreceptors, retina and eye cup. Nature Reviews. Neuroscience 8(12): 960–975.

Land, M. F. and Nilsson, D-E. (2002). Animal Eyes. (Willmer, P. and Norman, D. (eds)). Oxford: Oxford University Press.

Nathans, J. (1999). The evolution and physiology of human color vision: Insights from molecular genetic studies of visual pigments. Neuron 24(2): 299–312.

Oakley, T. H. and Pankey, M. S. (2008). Opening the ‘‘black box’’: The genetic and biochemical basis of eye evolution. Evolution Education and Outreach 4: 390–402.

Plachetzki, D. C. and Oakley, T. H. (2007). Key transitions during animal eye evolution: Novelty, tree thinking, co-option and co-duplication. Integrative and Comparative Biology 47: 759–769.

Salvini-Plawen, L. V. and Mayr, E. (1977). On the Evolution of Photoreceptors and Eyes. New York: Plenum Press.

Spudich, J. L., Yang, C. S., Jung, K. H., and Spudich, E. N. (2000). Retinylidene proteins: Structures and functions from archaea to humans. Annual Review of Cell and Developmental Biology 16:

365–392.

Terakita, A. (2005). The opsins. Genome Biology 6(3): 213. Yokoyama, S. and Yokoyama, R. (1996). Adaptive evolution of

photoreceptors and visual pigments in vertebrates. Annual Review of Ecology and Systematics 27: 543–567.

Glossary

Aniridia – Congenital condition resulting in the underdevelopment or lack of an iris.

Anophthalmia – Congenital absence of one or both eyes.

Ectopic eye – An eye that has formed in an abnormal location (an additional eye).

Eye field – A region of the anterior neural plate of vertebrate embryos that is fated to generate the eyes.

Gene homology – Two genes are homologous if they are derived from the same gene in a common ancestor.

Hypomorphic mutant – A mutation in which the altered gene product has reduced activity or in which the wild-type gene product is expressed at a reduced level.

Iris – Colored portion of the eye surrounding the pupil.

Microphthalmia – Congenital defect resulting in small eyes.

Retina – Light-sensing part inside the inner layer of the eye. The vertebrate retina consists of seven major cell classes.

Discovery and Structural Features

of Eye-Field Transcription Factors

Pax6

Vertebrate Pax6 (paired box gene 6) was identified, in 1991, in mouse and humans as a member of a multigene family of paired-box-containing (Pax) genes. Small-eye (Sey) mutant mice were found to have mutations in the Pax6 gene predicted to interrupt gene function. The Drosophila melanogaster (fly) mutant eyeless (ey) was subsequently shown to be homologous to vertebrate Pax6. Remarkably, the mouse Pax6 was shown to functionally rescue the eyeless phenotype in flies, demonstrating functional conservation of Pax6 from flies to mammals. Pax transcription factors contain a paired domain and either a partial or complete (as in the case of Pax6) homeodomain (see Figure 1 and Table 1 for EFTF structure and characteristics). In addition, Pax6 contains a proline–serine–threonine-rich (PST-rich) C-terminus.

The paired domain is an approximately 126-residue DNAbinding domain originally described in the Drosophila gene of the same name. Two distinct DNA-binding domains are present in the paired-type domain in the form of N-terminal and C-terminal subdomains. Alternative splicing of the Pax6 gene results in alternate isoforms with distinct DNAbinding activities with functional consequences. The 60-residue homeodomain forms a helix-turn-helix structure at the carboxy-terminal end that also binds DNA. As with the paired domain, an altered homeodomain can result in developmental defects. The PST-rich region acts as a trans-activating domain with binding sites for other proteins such as transcriptional coregulators. Pax6 mutants that truncate the C-terminal-half of the protein remove the PST-rich region, retain their DNA-binding capacity, but act dominantly to repress normal Pax6 function.

Six3

The first Six (Sine oculis homeobox) family member was identified in 1994 as the gene altered in the Drosophila mutant sine oculis (so), the most striking phenotype of which is the lack of eyes. Vertebrate Six3 (sine oculis homeobox homolog 3) was first identified in mouse in 1995 and shown to share significant amino-acid homology with so. Members of the Six family of homeobox transcription factors are characterized by two conserved domains – the Six domain (SD) followed by a more carboxy-terminal homeodomain (HD). The Six domain is typically 115 residues and is required for protein–protein interactions between Six genes and their binding partners. The homeodomain is a 60-residue DNA-binding domain (as described above). Both the SD and HD are required for normal function of the protein. The more recent identification of additional Drosophila Six family members also demonstrated vertebrate Six3 (and section titled ‘Six6’) share greater sequence homology with fly Optix than its namesake sine oculis. Based on sequence homology, intron–exon boundries, and the proteins with which they form complexes, Six family members are divided into three subfamilies. Six3 and Six6 have been assigned to the Six3/6 subfamily.

Six6

A second Six gene structurally related to Six3 was identified, in 1998, in zebrafish, chicken, and mouse as Six6 (sine oculis homeobox homolog 6) and Optx2 (optic six gene 2). Among the family of Six genes, Six6 shares greatest sequence homology and expression pattern with Six3,

suggesting they may have arisen via gene duplication. Despite their similarities, Six6 and Six3 can be easily distinguished at the amino-acid level. The most highly divergent regions lie at the aminoand carboxy-terminals to the Six and homeodomains. In particular, the region of Six6 amino-terminal to the Six domain is dramatically smaller than the corresponding region of Six3. For example, only nine residues are present at the amino-terminal to the Six domain in mouse Six6, while 88 residues are present in the Six3 protein.

Rax

Vertebrate Rax (retina and anterior neural fold homeobox) was identified, in 1997, by three independent groups in Xenopus, mouse, and zebrafish, and is shown to be expressed in the developing eyes and required for normal mouse eye formation. Rax – like Pax6 – is classified as a member of the paired homeobox class of transcription factors. In contrast to Pax6, however, Rax contains a paired-like homeodomain, but no paired domain. Within

Figure 1 Schematic illustrating the location of structural domains in the mouse eye-field transcription factors. The EFTFs – with the exception of the Six genes Six3 and Six6 – have distinct domain structures and are members of different transcription-factor families. Five of the seven bind DNA targets via a homeobox domain (HD). While Tbx3 (via the T-box) and Nr2e1 bind DNA through DNA-binding domains (DBDs) specific to their transcription factor family. Each EFTF also contains protein motifs that allow it to complex with other proteins. These protein–protein interactions serve to facilitate (or sometimes block) the ability of the transcription factor to bind its DNA targets. Co-regulatory proteins that bind to these regions can serve as co-activators or co-repressors to increase or decrease, respectively, the rate of expression from a target gene. Courtesy of Andrea Viczian.

the amino-terminal, Rax contains an octapeptide motif consistent with this homeobox gene subfamily. In the carboxy-terminal, a 15-residue region present in other paired-like genes termed the paired tail or OAR (otp, aristaless, and rax) domain is also conserved. Finally, the sequence carboxy-terminal to the paired-like homeodomain is PST-rich. These regions (octapeptide, OAR, and PST-rich) may function as transactivation domains.

Lhx2

First identified in rat as LH-2 (LIM Hox gene 2) in 1993, vertebrate Lhx2 (LIM homeobox-2) is a member of a large LIM-domain (Lin 11, Isl-1, Mec-3)-containing family of genes with diverse structure and biological functions. Mouse Lhx2 was subsequently shown to be required for normal eye formation. The LIM domain (two of which are found in Lhx2) is a cysteineand histidine-rich zincfinger motif involved in protein–protein interactions. The LIM domains of Lhx2 protein are located in the aminoterminal region and followed by a carboxy-terminal, highly conserved, DNA-binding homeodomain. There is little evidence that LIM domains of the LHX class can bind DNA directly, but may regulate DNA binding of the protein via interactions with other proteins.

Tbx3

Vertebrate Tbx3 was identified, in 1994, in mouse as a member of a large gene family characterized by a shared T-box protein motif. Mutation of the founding member of this family caused truncated tails in mice and was shorthand named T for short-tail. Once the gene was identified, the DNA-binding region of the T protein (which is 180–190 residues and can be located at any position in the protein) was subsequently named the T-box. The more than 50 T-box family members identified have been classed into five subfamilies. Tbx3 is a member of the Tbx2 subfamily that includes Tbx2-5 and is most similar to Tbx2.

Nr2e1

Vertebrate NR2E1 (nuclear receptor subfamily 2, group E, member 1) was originally identified in 1994 as Tlx,