Transcription factor genes and the developing eye: a genetic perspective

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1 1996 Oxford University Press Human Molecular Genetics, 1996, Vol. 5 Review Transcription factor genes and the developing eye: a genetic perspective Carol Freund 1, D. Jonathan Horsford 1,2 and Roderick R. McInnes 1,2,3,4, * 3 Program in Developmental Biology, Departments of 1 Genetics and 4 Pediatrics, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8 and 2 Department of Medical Genetics and Microbiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada Received June 20, 1996 We review the current knowledge of transcription factors in mammalian eye development. The 14 transcription factors presently known to be required for eye formation are examined in some detail, incorporating data from both humans and rodents. Aspects of the biochemistry, expression patterns, genetics, mutant phenotypes, and biological insights acquired from the examination of loss-of-function mutations are summarized. The other 32 tissue-restricted transcription factors that are currently known to be expressed in the developing or mature mammalian eye are tabulated, together with the timing and site of their ocular expression; the requirement for most of these genes in the eye is unknown. Contributions to mammalian eye development from the study of the genetics of the Drosophila eye are discussed briefly. Identification of the entire cohort of transcription factors required for eye development is an essential first step towards understanding the mechanisms underlying eye morphogenesis and differentiation, and the molecular basis of inherited eye abnormalities in humans. INTRODUCTION Genetic disease and the developing eye Geneticists have long been attracted to the study of the eye, mainly because they can see it. The retina in particular has long been recognized as an approachable part of the brain (1). This accessibility exists even in utero, and has enabled the use of the eye as a model system for the study of development in many organisms (2,3). In addition, detection of the effects of developmental aberrations on the eye is facilitated by the fact that the mature organ is well characterized at the biochemical, physiological and structural levels. Moreover, a large number of human and murine eye mutations are available for genetic and biological analysis because (i) the eye is a non-essential organ in man and other mammals, (ii) mutations affecting the eye often create readily recognized phenotypes, and (iii) the importance of normal vision to humans means that virtually any significant dysfunction of the eye is brought to clinical attention. In a 1985 survey of McKusick s Mendelian Inheritance in Man (4), the eye was involved in 27% of the 2811 phenotypes recorded, making it the fourth most common system affected by genetic disease in man (after the integument, the rest of the nervous system, and muscle). More than 50 loci for genetic ocular disease have been mapped in man (5), and more than 110 eye mutants have been described and mapped in mouse (6). In both species, many other phenotypically-defined inherited eye disorders remain to be assigned to loci. In this review, we summarize much of the available information on the genetics and eye phenotypes associated with mutations in genes encoding transcription factors in rodents and humans. At present, the genes encoding 14 transcription factors, belonging to six different protein families, have been shown to be required for the normal development of the mammalian eye. These genes are presented in Table 1, together with the pattern of inheritance of the disease phenotype and a brief description of the eye abnormalities seen with null alleles. A more detailed presentation of this information is made in the text. Although the study of these genes and their mutations has provided important clues about their roles in eye development, we presently know little about the mechanisms by which they exert their developmental effects. However, some of the principles underlying development in general, and of the eye in particular, are now well established. Essential features of eye and retinal development The eye develops from the embryonic forebrain in a series of distinct morphological stages that are depicted in Figure 1 (2). The pattern of expression of genes during eye development is generally reported in terms of these stages. A detailed discussion of the developmental events represented in Figure 1 is beyond the scope of this review, but since mutations in several transcription factor genes have been shown to affect the proliferation and/or differentiation of the neuroretina, some description of retinal development is required [reviewed in (3)]. Development of the neuroretina is first characterized by the proliferation of progenitor cells (Fig. 1). Any mitotic progenitor cell appears to have the potential to form any of the six major classes of mature neurons (photoreceptors, bipolar, horizontal, interplexiform, amacrine and ganglion cells), as well as Müller cells, a type of glial cell. *To whom correspondence should be addressed

2 1472 Human Molecular Genetics, 1996, Vol. 5, Review Table 1. The human or rodent eye phenotypes resulting from mutations in transcription factor genes. The phenotypes listed are those associated with null or putative null alleles. Phenotypes are discussed in more detail throughout the text. gene targeted knock out mice Pax6/PAX6 Pax2/PAX2 Chx10 Pou4f2 Hes1 mi Rxrα,β,γ Rarα,β,γ Gli3 Hfhbf1 Humans - Autosomal dominant with many alleles. - Phenotype: variable, may include aniridia and other anterior eye defects including cataracts, corneal defects; hypoplasia of ciliary body and fovea. Mouse/ Rat - Small eye (Sey) mouse/ rat. Semidominant mutation with several alleles - Phenotype: heterozygotes have small eyes, homozygous mutants lack eyes and nasal structures. Humans- Autosomal dominant renal-coloboma syndrome or autosomal dominant renal abnormalities. Mouse- Kidney, retinal defects (Krd ) mouse. Autosomal dominant. - Phenotype: thin retina, reduced retinal cell number, malfunction of photo-receptors and bipolar cells identified by electroretinography. Mouse- Ocular retardation (or) mouse. Autosomal recessive mutation. - Phenotype of or J allele: microphthalmia, optic nerve aplasia, thin hypocellular retina and a lack of differentiated bipolar cells. Mouse- Autosomal recessive null allele. - Phenotype: 70% reduction in ganglion cell number, small optic nerve, and thin outer and inner nuclear layers in retina. Mouse- Autosomal recessive null allele. - Phenotype: decreased retinal size, increased bipolar cell death, increased number of rods and amacrine cells and premature retinal differentiation; lens and corneal defects. - Overexpression phenotype: neuroretinal progenitor cells fail to differentiate in vitro. Mouse- Autosomal dominant and recessive, many alleles. - Phenotype: microphthalmia, retinal degeneration, colomboma and hypo-pigmentation of pigment layer. Mouse- Rarα autosomal recessive null allele. - Phenotype: ventral eye abnormalities (small retina, corneal defects, reduced or absent anterior chamber and optic nerve colomboma). - Paired Rarα, β and γ and Rxrβ autosomal recessive null alleles. - Phenotypes: defects of the eyelids, cornea, conjuctiva or anterior chamber; lens defects including abnormal lens fibres, retrolenticular membrane, ventral rotation; retinal defects including ventral retinal reduction, retinal dysplasia, colomboma; colomboma of the optic nerve. Mouse- Extra toes(xt ) mouse. Autosomal recessive. - Phenotype of Xt J allele: mild eye defects. Mouse- Autosomal recessive null allele. - Phenotype: ventrally rotated ellipsoid eyes, defective nasal retina with an irregular neuroepithelial surface. The multipotent neuroretinal progenitor cells of the optic cup first begin to leave the cell cycle at about embryonic day 10.5 (E10.5) in mouse (7), and therefore, as indicated in Figure 1, some retinal cell progenitors are continuing to proliferate at the same time as others are differentiating into mature retinal neurons. Each retinal cell type is born within a time frame characteristic of that cell; ganglion cells, for example, are amongst the first born retinal neurons in mouse ( born referring to the day a cell becomes post-mitotic, see below), and bipolar cells amongst the last. The timing and mechanisms of the commitment of a retinal progenitor to become a specific cell type are largely unknown, but it appears that progenitors are competent to become committed to a specific fate only after they exit the mitotic cycle (3). The commitment of a competent post-mitotic retinal progenitor cell to a specific cell fate is controlled by signals from the microenvironment. Identification of the environmental cues necessary to initiate the differentiation pathways for each specific retinal cell type is in its infancy (3). Nevertheless, it is clear that the cornerstones of developmental regulation in the eye as in other tissues are the signaling events within and between cells, and the endogenous control by transcription factors of the competence of cells to respond to signals. Thus, transcription factors may (i) make post-mitotic retinal cells competent to respond to appropriate environmental stimuli, and (ii) initiate the production of signals by the cell that modify the microenvironment, thereby influencing the developmental program of neighbouring cells (3). Therefore, in a particular environment, the combination of transcription factors expressed by a cell can be regarded as its transcription factor code. Thus, one biological role of transcription factor genes in development is that their expression often confers identity on a cell or a region of the embryo, i.e. transcription factors tell a cell what kind of cell it should be, or inform cells in a region about their location in the developing embryo (8,9). In both invertebrates and vertebrates, one well-known example of such regional specification occurs in the development of axial structures, which are defined by the particular combination of one class of homeobox genes the Hox genes expressed by cells in each region of the axis. The specific combination of Hox genes required for the formation of a particular region is referred to as the Hox code of that region (10,11). Recent exhaustive studies of the cis-regulatory control elements of two sea urchin genes show that a combinatorial set of several different regulatory modules control the temporal and spatial expression pattern of these genes (12,13, discussed by 14). Each module contains one or two unique transcription factor binding sites, in addition to sites for factors that bind within other modules as well. Each module acts as a positive or negative regulator of

3 1473 Human Nucleic Molecular Acids Genetics, Research, 1996, 1994, Vol. Vol. 5, 22, Review No Figure 1. A schematic presentation of early stages of mouse eye development, and their chronology during embryogenesis. The emphasis is on the morphogenesis of the eye cup and lens, and the development of the neuroretina and the retinal pigment epithelium (RPE). The first morphological manifestation of mammalian eye development is the evagination of the embryonic forebrain to form the optic pit. This evagination becomes a lateral diverticulation of the forebrain, the optic vesicle. As the optic vesicle approaches the overlying surface ectoderm, it induces the formation of the lens placode, which later invaginates to form the lens vesicle and, eventually, the lens. The cells of the lens vesicle nearest the optic cup differentiate into elongated lens fibers, while the lateral cells, closest to the surface ectoderm, remain as a monolayer. The invagination of the optic vesicle creates the two-layered optic cup, the outside layer giving rise to the RPE, and the inner multicellular layer of progenitor cells forming the neuroretina. The iris develops from the peripheral edge of the optic cup, from a cell layer continuous with the neuroretina. The cornea develops from a layer of overlying ectoderm. In the depiction of an E11.5 retina (above), some post-mitotic cells are illustrated, particularly ganglion cells. In the adult retina, the nucleus of each cell type is situated in one of three nuclear layers; the nuclear layers are separated by two plexiform layers containing the processes of retinal cells. The photoreceptor and ganglion cell layers contain those two respective cell types. The inner nuclear layer contains the cell bodies of four major cell types: horizontal cells, bipolar cells, amacrine cells, and Müller cells. PRL, photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. [Stages drawn from Kaufman (234), mature retina adapted from Dowling (1).] gene expression, or to amplify the level of expression. The correct combination of all of these modules is sufficient to recapitulate wild-type expression of a reporter construct in sea urchin development. Consequently, a major goal in the study of eye development will be to identify the transcription factors and cis-regulatory modules required for eye formation. Transcription factor genes active in development may also continue to be expressed in mature tissues, where they participate in the maintenance of the differentiated state (15). The few documented examples of this process in the nervous system include the requirement for the mec-3 homeodomain protein in sensory neurons of C. elegans (16,17), and the need for the microphthalmia gene product, a bhlh-zip transcription factor (see below), in the maintenance of the adult mouse retina (18). HOMEODOMAIN PROTEINS Homeodomains are 60 amino acid triple α helical motifs that bind to DNA (19) and are encoded by 180 bp sequences called homeoboxes. An excellent monograph and several reviews of homeobox genes have recently been published (9,20 23). More than 25 classes of homeodomains have been distinguished on the basis of their amino acid sequences (Bürglin, in 9), and the number of classes continues to increase; the newest additions can be found on the Internet (24). Other sequence motifs are often present in homeodomain proteins, including, for example, the paired, CVC, and POU domains, each of which are discussed below. These other motifs are generally thought and have sometimes been demonstrated to participate in protein protein interactions or DNA binding. More than 300 homeobox genes have been discovered (9,25) although to date, mutations that lead to genetic disease in man have been found in only eight: PAX2 (which has only a partial homeodomain, see below), PAX3 in Waardenburg Syndrome Types 1 and 3 (reviews: 26,27), PAX6 (see below), MSX2 in craniosynostosis (28), EMX2 in schizencephaly (29), PIT1 in hypopituitarism (30), POU3F4 in X-linked mixed deafness (233) and HOXD13 in synpolydactyly (31). Only PAX2 and PAX6 are associated with inherited structural defects of the eye in man, but mutations in two other homeobox genes, Chx10 (32) and Pou4f2 (33), lead to abnormal retinal development in mouse, and are therefore candidate genes for human eye defects as well. The PAX proteins The prd class of homeobox genes is composed of nine members in both man and mouse, identified as PAX or Pax genes, respectively. As indicated above, mutations in only PAX2/Pax2 or PAX6/Pax6 are associated with developmental abnormalities of the eye (34). Proteins of the prd class are defined by having a paired domain, an evolutionarily conserved 128 amino acid DNA binding motif named after the Drosophila gene, paired, in which it was first recognized (35). The nine PAX genes can be divided into four classes (34), depending on whether the gene also encodes, in addition to a paired domain, a paired homeodomain and/or an octapeptide: Class I genes (PAX1 and PAX9) have an octapeptide but do not have a homeobox; Class II genes (PAX3 and PAX7) have both elements; Class III genes (PAX2, PAX5, and PAX8) lack an octapeptide and have only a partial homeobox; and Class IV genes (PAX4 and PAX6) have a complete homeobox but

4 1474 Human Molecular Genetics, 1996, Vol. 5, Review lack an octapeptide. Both the paired domain (36) and the paired-type homeodomain (37,38) have DNA binding activity, but the function of the octapeptide is uncertain it may participate in protein-protein interactions (39). All of the Pax genes are expressed in the developing embryo, and all but Pax-1 and Pax-9 are expressed in the developing nervous system (40,41). In addition to eye phenotypes that result from PAX2 and PAX6 mutations, loss of functional alleles of Pax1, PAX3/Pax3, or Pax 5 are associated with mutant phenotypes in organs other than the eye, in either man or mouse. The PAX6/Pax6 gene A dramatic illustration of the centrality of homeobox genes in the specification of cell types and body regions comes from the work of Gehring and his colleagues, who showed that ectopic expression of eyeless, the Drosophila PAX6 orthologue (42), causes development of morphologically normal eyes on the wings, legs, and antennae (43). This work illustrates powerfully not only the apparent role of the eyeless-pax6 gene as a master regulator of Drosophila eye development, but also the incredible evolutionary conservation of developmental mechanisms in the higher eukaryotes (44). PAX6 is also required for eye formation in mammals, since mice with a Pax6 / genotype fail to develop eyes at all (45 48). In humans, the importance of PAX6 in selecting cells or defining regions of the eye is illustrated by the abnormalities of anterior eye structures (such as aniridia) seen in patients with haploinsufficiency of PAX6. Greater attention is given PAX6 in this review than any other gene, because with respect to the eye, there is much better understanding of its biology and genetics than of any of the other proteins discussed (Scheme 1). Scheme 1. Human PAX6 protein structure. Indicated are the locations of the paired domain (PRD), the paired type homeodomain (HD) and the alternative splice (arrowhead). Developmental expression of Pax6. Pax6 is expressed during mouse embryogenesis in parts of the spinal cord, hindbrain, and forebrain, including the anterior pituitary, olfactory pit, and developing eye (49,50). Pax6 expression is first evident in the eye at E8.0 in the optic pit (Fig. 1). By E9.5, expression is seen in the neural epithelium of the optic vesicle, the optic stalk, and the prospective lens ectoderm. The gene continues to be expressed in all these cell types as the optic cup and the lens vesicle form between E10 and E12 (Fig. 1). On day E12.5, cells of the corneal ectoderm, the lens, and inner layer (neuroretinal progenitors) of the optic cup maintain Pax6 expression, while expression decreases in the outer layer of the optic cup (future pigmented epithelium) as the cells become post-mitotic. In later stages expression is still detected in the actively proliferating cells of the neuroretina, lens and cornea, as well as in mature (i.e. post-mitotic) cells of the ganglion cell layer. Interestingly, transplantation experiments in the developing rat eye demonstrate that, at least in early eye morphogenesis, the Pax6 gene product is absolutely required only for the development of the prospective lens ectoderm, and not in the optic vesicle (where it is also expressed) (51,52). It should be noted, however, that this finding does not exclude the possibility that Pax6 expression is essential in later stages of retinal development, such as ganglion cell differentiation. The PAX6 protein and the regulation of target gene expression. Pax6, or its orthologues in other species, acts as an important stimulatory factor for the expression of several lens crystallin genes: chicken (53) and mouse (54) αa-cry, mouse βb2-cry (55), chicken δ1-cry (54), and guinea pig ζ-cry (56). The quail homologue, Pax-QNR, has been shown to autoregulate (57). There is also evidence from in vitro DNA binding assays that Pax6 can bind to the neural cell adhesion molecule L1 promoter region (58). A splice variant of Pax6 causing the inclusion of 14 amino acids in the paired domain was recognized in the original cdna clone (49), confirmed in the genomic clone (59), and shown to be evolutionarily conserved (60). The binding affinity of Pax6 is altered dramatically by the inclusion of this alternative exon, exon 5a (61). Normally both isoforms are expressed in eye, brain, spinal cord, and olfactory epithelium, although the 5a insertion form is less abundant (61). Preliminary examination of the binding affinity of the two PAX6 isoforms to the promoter sequence of βb2-cry shows that each isoforms binds to distinct regions (55). Regulation of Pax6 activity may also occur at the transcriptional level. Studies of the quail Pax-QNR show two possible promoters, one used in cells which differentiate non-neuronally, and both used in the developing neuroretina (2). A neuroretina specific enhancer is located 7.5 kb downstream of the neuronal specific promoter (63), and binding sites for c-myb, a transcriptional activator of Pax-QNR, have been identified (64). There are certainly more features of Pax-QNR regulation to be determined, because as Plaza points out, mice with no c-myb expression (65) do not show a mutant phenotype in the tissues where Pax6 is expressed (64). Mutations in the PAX6/Pax6 genes. Pax6 mutations cause malformation of the eye in several species: in humans a variety of clinical phenotypes, including aniridia (66), isolated foveal hypoplasia (67), Peters anomaly (68), anophthalmia (69), autosomal dominant keratitis (70) as well as a unique ocular syndrome (61); in mice (47) and rats (71), the Small eye phenotype; and in Drosophila, the eyeless phenotype (42). In mice, Small eye (Pax6 Sey ) is a semidominant mutation which maps to chromosome 2 (45). Heterozygotes (Pax6 Sey /+) have small eyes, and homozygous (Pax6 Sey /Pax6 Sey ) completely lack eyes and nasal structures (45,46,48). The rat phenotype (rat Small eye, rsey) is very similar (51). Four alleles of Small eye have been identified in mice, Small eye (Pax6 Sey ), Small eye Dickie (Pax6 Sey-Dey ), Small eye Harwell (Pax6 Sey-H ), and Small eye Neuherberg (Pax6 Sey-Neu ). Pax6 Sey, a nonsense codon truncates the protein before the homeodomain, and Pax6 Sey-Neu, a splicedonor mutation of exon 10, have similar phenotypes, while Pax6 Sey-H, a large deletion, has a more severe phenotype in that homozygotes die either before or shortly after implantation (47). Mice with the Pax6 Sey-Dey mutation also have a severe phenotype, in that homozygotes die early in pregnancy, and heterozygotes are of reduced body size, with small eyes, coloboma, small or absent

5 1475 Human Nucleic Molecular Acids Genetics, Research, 1996, 1994, Vol. Vol. 5, 22, Review No lens, abnormal folding of the retina, and reduction of the RPE, and usually the anterior chamber is absent (72). Many of the human phenotypes associated with mutations in PAX6 result from errors in the development of the anterior segment of the eye. PAX6 was originally identified as the AN gene, because it was positionally cloned from the Aniridia region (66). The aniridia phenotype is characterized by the partial or total absence of the iris, and is often associated with other abnormalities such as cataracts, corneal vascularization and opacification, glaucoma, and hypoplasia of the ciliary body and retina. Aniridia is a feature of the WAGR syndrome (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation) which is localized to chromosome 11p13 (73). Hanson and van Heyningen (74) have recently catalogued the various other mutations in PAX6 that result in Aniridia. These mutations include seven splicing errors, nine insertions or (internal) deletions, eight nonsense mutations and only two missense mutations (one missense mutation, R26G, actually caused Peters anomaly; see below). Since that review, additional novel mutations causing aniridia have been reported, including two more nonsense and one splicing error (75). Fantes (76) reports two very interesting aniridia pedigrees with chromosomal rearrangements involving 11p13, in which there are no mutations in the PAX6 coding sequence, and apparently no splice errors. The chromosomal breakpoint in both cases is greater than 85 kb distal to the 3 end of PAX6, suggesting a position effect (76). Mutations in PAX6 also cause Peters anomaly (68), which presents with corneal defects including central corneal opacity and adhesions between the lens and cornea, sometimes including the iris. One individual had a typical WAGR deletion, encompassing PAX6, but the phenotype is much more similar to Peters anomaly than aniridia. The other carried a missense mutation, R26G, which replaces a highly evolutionarily conserved residue and disrupts the paired box. Several additional eye phenotypes have been found to be associated with mutations in PAX6, adding to the difficulty of correlating specific clinical phenotypes with particular mutations. Autosomal dominant keratitis, characterized by foveal hypoplasia and corneal opacification and vascularization (70) was associated in a family with an exon 11 splice-acceptor site mutation; the resultant truncated protein is the same as that produced from the mouse Sey Neu allele. A unique ocular syndrome of bilateral juvenile cataracts, peripheral corneal opacification, glaucoma, and pendular nystagmus was found to result from a PAX6 mutation that alters the ratio of the two splice variants of PAX6 (61). These patients were unlike usual cases of aniridia in that their irides were largely normal, with only minor abnormalities in the anterior portion of the iris (61). In contrast to the majority of patients with PAX6 mutations, in which the anterior segment of the eye is affected, one family with isolated foveal hypoplasia and no anterior eye defects has recently been discovered. The phenotype in these patients resulted from a PAX6 missense mutation, R125C, which alters a conserved residue in the carboxy-terminal portion of the paired domain (67). Finally, a very severe phenotype, more closely resembling that of the homozygous mutant small eye mouse than any other PAX6 mutation, has been reported in a compound heterozygote with two nonsense mutations (69). This patient had no eyes, severe craniofacial defects, and other central nervous system abnormalities. Analysis of the genetics and biology of PAX6 has provided many insights into the mechanisms of eye development and of human eye developmental defects. The extreme variation in phenotypes suggests that modifier genes may well have a significant impact on the physical manifestations observed for a particular PAX6 mutation. A major question is whether the importance of Pax6 in vertebrates approaches the status of its eyeless orthologue in flies, as a master regulator of eye development. Although it is seems unlikely that the regulatory processes that coordinate the development of vertebrate eyes are as directly controlled by PAX6 as they are in Drosophila to the extent that ectopic PAX6 expression in mammals would lead to eye formation no data bearing on this issue are currently available. The PAX2/Pax2 gene The PAX2 protein (Scheme 2) is most similar to PAX5 and PAX8, based on sequence homology in the paired domain and organization of the structural gene (77). Scheme 2. Human PAX2 protein structure. Indicated are the locations of the paired domain (PRD), octapeptide (OCT) and homeodomain remnant (HD*). Developmental expression of Pax2. Expression of PAX2 in early development of mice and zebrafish is very similar, and is seen in the brain, optic stalk, auditory vesicle, developing kidney, and single cells in the spinal cord and the hindbrain (78 80). PAX2 expression is essential for proper kidney development, and aberrant expression has been detected in Wilms tumors (review: 81). At least two isoforms of the PAX2 protein are detected in kidney, derived from differentially spliced messages which maintain or disrupt the homeodomain (78,82), and differential splicing of a conserved portion of the 3 coding sequence which results in an alternative carboxy-terminus has been found (83). The significance of these isoforms and their presence in the eye is unknown. Pax2 expression in the eye is detectable by E9 in the optic vesicle and optic stalk, and is greatest in the ventral half of the optic cup. Pax2 is not expressed in the developing or mature lens (79,80). The PAX2 protein and the regulation of gene expression. Pax2 expression levels have been shown to be affected by several factors, including increased expression with NGF, decreased expression with BDNF (84), repression by WT1 (85) and activation by EGR1 (86). Studies of the zebrafish cyclops mutant (87) and vertebrate hedgehog proteins (88) have shown that the regulation of both Pax2 and Pax6 expression is influenced by signaling molecules (perhaps including Sonic hedgehog) in midline tissue. The PAX2 protein is able to bind to the promoters of p53 (34), EGR1 (81), and En-2 (89), but its role in vivo in general, and in the eye in particular, is still to be determined. Mutations in the PAX2/Pax2 genes. Mutations in PAX2 cause human and mouse developmental abnormalities. The gene maps to chromosome 10q24 in humans (90) (previously localized to 10q25 in ref. 91) and chromosome 19 in mouse (77). Mutations in the mouse Pax2 gene are associated with kidney and retinal defects in the Krd mouse (92). This mutation arose because of a transgene insertion that resulted in a 7 cm deletion spanning the

6 1476 Human Molecular Genetics, 1996, Vol. 5, Review Pax2 locus. Krd homozygous mutant mice die in early embryogenesis and heterozygotes have an overall thin retina with reduced numbers of cells in all neuroretinal layers, a normal RPE, and malfunction of the bipolar cells and photoreceptors as demonstrated by electroretinography. Although many genes are likely to be deleted in this mutant, the kidney and ocular phenotypes correspond well to the expression pattern of Pax2 (92). A perinatal lethal gain-of-function mutation was generated in transgenic mice by Pax2 expression driven by a human CMV-promoter (93). Newborn transgenic animals had ectopic Pax2 expression in heart, liver, lung, pancreas and gut, but these tissues appeared grossly normal. The eyes opened early (by E18), but the retina and lens were normal. The kidneys were reported to be the major site of abnormal histology. A null allele of Pax2 has been generated by homologous recombination, (94), but the eye phenotype has not been reported. The mouse phenotypes associated with Pax2 mutations suggested the candidate human phenotypes that might arise from mutations in the human gene. Sanyanusin and colleagues were able to identify families with two distinct dominant phenotypes, renal-coloboma syndrome (95) and autosomal dominant renal anomalies, vesicoureteral reflux and optic nerve colobomas (96). The mutations in both families were putative null alleles. The mutation found in the family with renal anomalies, vesicoureteral reflux, and optic nerve colobomas is predicted to delete most of the octapeptide and the remaining carboxy-terminal portion of the protein (96), while the mutation found in the family with renal-coloboma syndrome removes most of the paired domain and all sequences towards the carboxy-terminus (95). There is a significant difference in the phenotypes expressed in these two families, but also a great deal of variance in the phenotype within a family. As in the Krd mice (92), the differences in the severity of the phenotype may be due to the genetic background on which the mutation is expressed. The Chx10 homeobox gene The paired class homeodomain of CHX10 is 82% identical to that of the nematode protein, CEH-10 (97), and both are members of the paired class of homeodomain proteins (98). An additional 54 amino acid motif, called the CVC domain (Scheme 3) (99), is located immediately carboxy-terminal to the homeodomain, and is named after the first three proteins in which it was identified: CHX10 (98), VSX1, a goldfish retinal protein, (100), and CEH-10 (97). Although the function of the CVC domain is unknown, the fact that its sequence is 70% conserved between CHX10 and CEH-10 suggests that this motif has some critical role, perhaps in protein-protein association. Scheme 3. Mouse CHX10 protein structure. Indicated are the diverged octapeptide (OCT*), the paired-like homeodomain (HD) and the CVC domain (CVC). Developmental expression of Chx10. In the mouse, Chx10 is most abundantly expressed in the developing and mature retina, although transcripts are also seen in regions of the developing thalamus, hindbrain, and ventral spinal cord. The earliest known expression of Chx10 is in the developing eye at E9.5, where transcripts are detected throughout the optic vesicle in cells which later give rise to the neuroretina. As the optic cup forms, Chx10 expression continues in the neuroretinal progenitors of the inner layer of the optic cup, but is not observed in the developing or mature retinal pigment epithelium. As the retina differentiates, Chx10 expression becomes restricted almost entirely to the inner nuclear layer (Fig. 1), where it is found most abundantly in the bipolar cell interneurons (32,98). Thus, the formation of the photoreceptor and ganglion cell layers (Fig. 1) is associated with the loss of Chx10 expression in the cells of those layers, raising the possibility that the extinction of Chx10 expression may be required for the differentiation of those layers (98). This expression pattern suggests that Chx10 may be important in the retina at three different times: (i) early, in undifferentiated progenitors; (ii) later, during retinal differentiation; and (iii) in maturity, in the non-mitotic neuroretina, particularly in bipolar cells. It is notable that ceh-10 is expressed in an interneuron called AIY which, like the bipolar interneurons in which Chx10 is expressed, also receives synaptic input from a sensory cell. In the case of C. elegans, the AIY cell receives input from a thermosensitive cell called AFD, and in the mammalian case, the bipolars receive input from the photoreceptors. These findings suggest that there has been a conservation of sensory regulatory mechanisms in evolution (99). A Chx10 null allele in the ocular retardation mouse. Genetic analysis has demonstrated that Chx10 is required for the development of the mouse eye. Both Chx10 and a recessive mutant called ocular retardation (or) map to the same region of chromosome 12, a coincidence that led to the discovery that mice carrying the or J allele have a premature stop codon in the Chx10 homeobox (32). Ocular retardation mice have numerous developmental eye defects, including microphthalmia, a thin hypocellular retina, optic nerve aplasia, reduced proliferation of neuroretinal progenitors that can be detected as early as E10.5, and a specific absence of differentiated bipolar cells. It is unclear whether the absence of detectable bipolar cells in the mature retina reflects a failure to specify these neurons during development, or impaired differentiation of correctly specified cells. Nevertheless, this cellular defect is specific, since all the other major retinal cell types are present and correctly positioned (32). The defect in or J neuroretinal cell proliferation is unlikely to be primarily responsible for the failure to make differentiated bipolar cells, since Müller cells, the other cell type born at the same time as bipolar cells (7), are present in the mutant retina (32). To date, no human phenotype has been associated with mutations of CHX10. Elucidation of the precise role of CHX10 in mammalian eye development will require identification of the target genes of the CHX10 transcription factor, which may differ according to the time and stage of development. The similarity of the murine ocular phenotypes that result from the loss of function of Chx10 and of another transcription factor gene, Hes1, is discussed below. BRN3B/Pou4f2 The POU homeodomain proteins are named for the first members of the class that were identified Pit-1/GHF-1, Oct-1 and Oct-2 (mammalian), and Unc-86 (nematode) (101). They contain a POU domain which consists of three components: the conserved POU specific domain of approximately 70 amino acids, and a

7 1477 Human Nucleic Molecular Acids Genetics, Research, 1996, 1994, Vol. Vol. 5, 22, Review No POU class homeodomain of 60 amino acids, connected by a linker of variable size. Many POU domain proteins recognize an octamer DNA sequence (102), and the presence of both the POU specific domain and the POU homeodomain is important for DNA binding and specificity (reviewed in 103). Both the POU specific domain (104) and the POU homeodomain (105) participate in protein-protein interactions. The PIT-1 POU protein has been shown to bind to a 28 base pair DNA element as a dimer (106). POU proteins have been classified into six families based on sequence homology of the POU specific domain and POU homeodomain. One group of class IV POU genes, Pou4f1, 2, and 3 (formerly referred to as Brn-3a, -3b, and -3c, or Brn-3.1, -3.2, and -3.3) contain an additional conserved 25 residue domain of unknown function, called the upstream homology domain (UHD) (Scheme 4). Drosophila daughterless (da) proteins (111). A conserved basic DNA-binding domain is situated next to two amphipathic α-helices separated by an interhelical loop. bhlh proteins act as either hetero- or homodimers in transcription. Studies of both mammals and Drosophila demonstrate that bhlh genes have important regulatory roles in myogenesis and neurogenesis (review: 112). Mutations in two bhlh genes, Hes1 and microphthalmia (which also possesses a ZIP domain, see below), are associated with abnormalities of mammalian eye development (18,113). The Hes genes The Hes genes were originally cloned as mammalian homologues of the Drosophila genes hairy(h) and Enhancer of split (E(spl)) genes (Scheme 5) (114). Scheme 4. Human BRN3B protein. Indicated are the POU IV specific upstream homology domain (UHD), the POU specific domain (POU) and the POU homeodomain (HD). Developmental expression of Pou4f genes. Several POU-domain transcription factors have been found to be expressed in the developing retina, particularly in ganglion cell development (see Table 2; review: 107). Expression of the Pou4f family is restricted to ganglion cells of the retina, somatosensory neurons in the dorsal root, trigeminal ganglia (108), spinal cord and brain stem (109). In the retina, different types of ganglion cells can be described based on morphological and physiological criteria (107). Retinal ganglion cells show an overlapping set of Pou4f1, 2, and 3 expression (108). In mouse, Pou4f1 and Pou4f2 are expressed together in approximately 70% of the ganglion cells, and a subset of these cells also express Pou4f3 (108). Mutation of the Pou4f2 gene. Gene targeting of Pou4f2 is associated with a loss of a subset of ganglion cells (33). Pou4f2 homozygous or heterozygous mutant mice show no obvious physiological or behavioural phenotype, and appear much like their normal littermates (33). However, in the eye, absence of Pou4f2 results in a reduction in the number ganglion cells by about 70%, a percentage of cells identical to that expressing Pou4f2. The reduction in cell number seems not to be due to increased cell death, but rather due to inadequate proliferation of ganglion cell precursors (33). Absence of these cells also results in a smaller optic nerve and thinner inner and outer nuclear layers, probably due to a reduction of potential synaptic connections in the retina (33). The human homologues of Pou4f1, 2, and 3 (called BRN3A, BRN3B, and BRN3C) have been cloned (108) and mapped to human chromosomes 13 (108), 4q31.2 (110), and 5 (108) respectively. No human phenotype has yet been associated with BRN3 mutations. BASIC HELIX-LOOP-HELIX PROTEINS A basic helix-loop-helix overview The basic helix-loop-helix (bhlh) motif was first identified in the murine immunoglobulin promoter binding proteins E12/E47 and was then noted to be present in the murine myc, MyoD and Scheme 5. Mouse HES1 protein, with the location of the basic Helix-Loop- Helix (bhlh) domain. There are several mouse or rat members of the Hes gene family (Hes1 through 5) (114,115) and one identified human homologue (HRY) (116). The latter maps to 3q28-q29 and is homologous with the rat Hes1 gene (only three amino acids differ). HES1 acts as a transcriptional repressor by binding to a specific target sequence, referred to as the N-box (114, 117). Hes1 and 5 are expressed in the developing mouse and rat (114, 115). Hes1 is expressed at high levels in mouse neuroretinal progenitors at the earliest time examined (E10.5). Expression continues until P0, after which it begins to decline to low levels at P3 7 (113), the time at which the final progenitor cells differentiate (7). Tomita et al. report that ganglion cells do not express Hes1, but no information was given on Hes1 expression in other differentiated retinal cells. Taken together, these findings are consistent with Hes1 acting as a negative regulator of neurogenesis (neural development) in the retina. Hes5 is also expressed as alternatively spliced transcripts in the developing rat eye (115) suggesting an additional level of complexity. A high level of Hes5 expression is detected in the outer nuclear layer at E18.5 (the first time point discussed), expression continues until P6, but by P14 expression is almost undetectable. The specific cell types expressing Hes5 in the developing eye have not been determined. Hes1 overexpression phenotypes in retinal cultures. Hes1 has been shown to be necessary for murine eye development by both over-expression and loss-of-function analyses (113). In retinal cultures from E17.5-P0 mice that are overexpressing Hes1 from retroviral infection, the retinal cells are undifferentiated, as indicated by a lack of mature retinal cellular morphology or marker expression. This undifferentiated neuronal phenotype is not unique to the retina it was also seen in the CNS from persistent expression of Hes1 from retroviral infection (118). Hes1 gene-targeted mice. In contrast to the phenotype resulting from increased Hes1 expression, mice lacking Hes-1 function exhibit premature retinal differentiation. These mice die at birth, probably from severe neural tube defects (119). Eye development is abnormal by E9.5, neuroretinal progenitors differentiate

8 1478 Human Molecular Genetics, 1996, Vol. 5, Review prematurely into ganglion cells and express neuronal markers earlier than the normal retina (113). By E10.5, the mice display small deformed optic cups with ventral openings and disturbed lens development. The eye phenotype is variable, in extreme cases mice completely lack a neural retina. By E lens and corneal structures are abnormal, as is the gross retinal morphology, for example lack of lamination and formation of rosette-like arrangements. Evidence from retinal cultures from Hes1-deficient mice also suggest that Hes1 is also critical in the differentiation and survival of specific retinal cell types (113). Retinal cultures from E17.5 Hes1 deficient mice fail to laminate and also display rosette-like structures. Rod photoreceptor differentiation is premature and non-uniform. Horizontal cell differentiate early, although the ganglion cells mature normally. The Hes1-deficient retinal progenitors form all retinal the cell types, but the cell ratios are skewed: the bipolar cells all die by day 14 of the culture, while the overall percentage of amacrine cells and rod photoreceptors is increased. Hes1 thus appears to be required for bipolar cell survival. Hes1 is also important for rod and amacrine development since lack of Hes1 seems to make these cells prematurely competent to respond to an external differentiation signal. The early commitment of retinal progenitors to rods therefore depletes the size of the total progenitor pool, producing a retina that is smaller overall. The role of Hes1 as a neural development inhibitor is conserved in Drosophila. A Drosophila homologue of the Hes genes is Enhancer of split [E(spl)]. This gene inhibits the proneural genes of the achaete scute complex (AS-C), which are involved in the decision to follow either a neural or epidermal cell fate (112). Hes1 seems to play a similar role in mammals in the inhibition of neurogenesis. One of the mammalian AS-C homologues- Mash1, is involved in neural-specific cell determination and is expressed in the eye, but it is not necessary for normal eye development ( ). Thus other mammalian AS-C homologues may exist and participate in eye development. The other Drosophila homologue of Hes1, hairy, inhibits Drosophila eye development by negatively regulating (along with the HLH gene extramacrochaete ) the bhlh proneural gene atonal (112,123). The role of Hes1 in inhibiting eye differentiation suggests that this inhibition may also be conserved in mammals, although the expression in the developing eye of Math1, the mammalian homologue of atonal, has not been examined closely (124). In addition, evidence suggests Hes1 may be a critical target of the Notch pathway. In Drosophila, E(spl) is a part of the highly conserved Notch receptor pathway which directs cells toward a non-neural cell fate (review: 125). An activated form of a Xenopus orthologue- Xotch, results in a phenotype in which cultured retinal cells maintain an undifferentiated state (126). Activated Notch expressed in chick retinal culture also inhibits ganglion cell differentiation (127). This phenotype is very similar to the one resulting from persistently expressed Hes1, implicating Hes1 as an important gene in the Notch pathway. Both Notch and Hes genes are expressed in the mammalian eye ( ), consistent with the conserved role of these genes in retinal development. Chx10 and Hes1 are required for bipolar cell development or maintenance. The similarity of the retinal phenotype of mice lacking Chx10 and Hes1 are noteworthy (32,113). Loss of either gene product causes a decrease in the number of bipolar cells. In null-allele or J mice lacking the CHX10 protein, bipolar cells are either not specified, or do not differentiate from committed progenitors. In contrast, bipolar cells develop in retinal cells cultured from Hes1-deficient progenitors, but do not survive. Chx10 and Hes1 may therefore be components of a developmental pathway that specifies maturation and survival of bipolar cells. Hes1 clearly plays a broader role in development, however, since the absence of Hes1 function impairs neurogenesis in general, while the effects of a loss of Chx10 function appear to be restricted to the eye. Double mutants may help unravel the position of these genes in the developmental network that controls retinal neurogenesis. MITF/Mitf The microphthalmia gene (Scheme 6) is another member of the basic helix-loop-helix family of transcription factors, and also contains a leucine-zipper domain, which is involved in proteinprotein interactions (132,133). The microphthalmia gene [human: MITF (134); mouse: Mitf (135,136)] encodes a protein similar to the bhlh-zip proteins TFEB, TFEC, and TFE3 (137). These four proteins, referred to as the MiT family, have been shown to heterodimerize in all combinations, each with varied DNA binding affinities (137). In addition to the potential modulation of MITF activity through various heterodimers, multiple isoforms are produced through differential splicing. There are two alternatives for exon 1 (138), as well as an alternative splice between exon 5 and 6, resulting in two forms which either include or omit six amino acids immediately amino terminal to the bhlh domain (135). The splice form with the 6 amino acid insert has slightly higher DNA binding affinity (137). Scheme 6. Human microphthalmia associated transcription factor (MITF). The basic Helix-Loop-Helix domain (bhlh) and the leucine zipper (ZIP) domains are indicated. The arrowhead shows the location of the alternative splice seen in the mouse homolog (mi). Note: mi also uses alternatively spliced amino termini. Embryonic tissue expression of Mitf is seen at E13.5 in the retinal pigmented epithelium, in individual cells surrounding the otic vesicle, and in hair follicles (135); a recent study of the MITF gene reveals the presence of a melanocyte-specific promoter (139). Examination of MITF activity as a transcriptional regulator has shown that MITF activates a reporter construct containing an M-box element, a conserved 11 bp sequence from the promoters of three major pigmentation enzyme genes (137). Two groups have examined the tyrosinase promoter and found no evidence of MITF binding in vitro, although it did activate transcription from the tyrosinase promoter in expression assays (140,141). The microphthalmia gene product also interacts with retinoblastoma protein in vitro (142). Mutations in the MITF/Mitf genes. Mouse Mitf maps to chromosome 6 (143). Numerous alleles of microphthalmia, which cover the mutational spectrum, cause both autosomal dominant and autosomal recessive disease (review: 18). Mitf mutations affect the cells in which the protein is developmentally

9 1479 Human Nucleic Molecular Acids Genetics, Research, 1996, 1994, Vol. Vol. 5, 22, Review No expressed: the retina, the ear and the hair, as well as other systems. The most severe phenotypic manifestations occur due to alleles in which the DNA binding domain is mutated. The range of phenotypic characteristics includes microphthalmia, retinal degeneration, osteopetrosis, inner ear defect with possible hearing loss, pigmentation disorders, and reduced mast cell numbers. An important feature of the severe eye phenotype is hyperplasia of the retinal pigment epithelium, with subsequent choroidal fissure closure problems. With some alleles, retinal degeneration is progressive and probably secondary to poor interaction between the RPE and photoreceptors (18). The human microphthalmia transcription factor gene, MITF, was cloned from a human melanocyte library based on homology to the mouse Mitf gene (134), and mapped to chromosome 3p14.1-p12.3 (134,136). Patients with Waardenburg Syndrome type 2 (WS2; OMIM ) have been found that are heterozygous for splice-site mutations that result in null alleles (144). The human disease is dominant, while the mouse diseases are generally recessive, with normal or only very mildly mutant phenotypes in heterozygotes. The striking microphthalmia seen in mice with homozygous Mitf mutations is absent from the phenotype of WS2, in which the only clinical eye features are heterochromic irides, hypoplastic iris stroma and albinotic fundus (145). The primary characteristics of WS2 are deafness, a white forelock, and absence of dysmorphic features. (WS2 stands in contrast to WS1 and WS3, which have facial dysmorphology and are due to mutations in the PAX3 gene.) It may be that there are numerous human phenotypes which result from MITF mutations, and not all appear as developmental defects. Vitiligo (progressive depigmentation) has been suggested as a human phenotype correlate of mouse microphthalmia (146). It will be important to examine some of the human retinal degeneration phenotypes for MITF mutations, since it is clear that in mice retinal degeneration is an associated feature. WINGED HELIX PROTEINS Hfhbf1 The winged helix is a DNA binding domain, named for its three-dimensional structure which consists of three alpha helices and two wing like loops that interact with the DNA (Scheme 7) (147). This motif was first identified in the HNF-3 family of transcription factors, and is related to Drosophila forkhead (review: 148). Scheme 7. HFHBF1 protein. The winged helix domain (WHD) is indicated. Developmental expression and mutation. Many WH genes are active in development, and at least two, Hfhbf1 and 2 (formerly Brain Factor 1, 2; BF1, 2) (149,150), are expressed in the developing brain and retina. Expression in the mouse neural retina begins at E9.5 for Hfhbf1 and E10.5 for Hfhbf2, and continues for both until at least E17, the last developmental time point examined (see Table 2). In the eye, the expression patterns of these genes are restricted such that Hfhbf1 is specific to the nasal optic stalk and retina, and Hfhbf2 is restricted to the temporal optic stalk and retina. Hfhbf1 is necessary for mouse eye development (151). Homozygous null Hfhbf1 animals show a striking ocular phenotype, with ventrally rotated ellipsoid eyes, and a defective nasal retina with an irregular neuroepithelial surface. It will be interesting to examine Hfhbf2 mutants for similar phenotypic features specific to the temporal retina. Human homologues, FKHL1, 2 [Forkhead (Drosophila)-like 1, 2 formerly BRAIN FACTOR-1, 2;BF-1, 2] have been identified and both mapped to 14q11 13 (152,153). Identification of possible human mutant phenotypes is an important next step. Elucidation of the downstream targets of these genes and the mechanisms underlying this dramatic phenotype will not only provide both insight into the specific roles of Hfhbf1 (and 2) in eye development but also into eye morphogenesis in general. ZINC FINGER PROTEINS The GLI/Gli genes The zinc finger motif was first identified in the Xenopus transcription factor TFIIIA (154,155) and can bind DNA or RNA. The GLI genes are related to the Krüppel family of transcription factor genes (156) which encode five zinc fingers, of which the last four contact DNA (157). GLI is so named because it was identified as an amplified gene in a human glioblastoma (158). Additional GLI genes have been cloned in human [GLI2 and GLI3 (159)) and mouse (Gli, Gli2 and Gli3 (160)] (Scheme 8). Scheme 8. Mouse BL13 protein. The domain which encodes the five zinc fingers is indicated (ZFD). Developmental expression and mutant phenotype. In the mouse, Gli, Gli2, and Gli3 are all broadly expressed in ectoderm and mesoderm beginning at E7.0-E7.5, and become more restricted as development proceeds (160). All three are expressed in the optic vesicle of the developing mouse eye at E9.5, and by E14.5, Gli is detected in the neural retina and optic stalk, while Gli2 and Gli3 are restricted to the optic stalk and the lens (160). The human GLI3 gene, which maps to chromosome 7p13 is mutated in the Greig cephalopolysyndactyly syndrome (GCPS) (161). GCPS does not show a specific developmental eye defect, but is characterized by an abnormal skull shape and malformed digits. A model for GCPS is the extra-toes mouse phenotype (162). At least two alleles, Gli3 xt and Gli3 xt-j, have ocular phenotypes, and both are due to Gli3 deletions ( ). Non-ocular features of mice with the Gli3 xt allele, an 80 kb deletion, include preaxial polydactyly in heterozygotes, while in homozygotes the effect is lethal, with severe skeletal and central nervous system deformities (163). The eye phenotype of homozygous Gli3 xt embryos at E10.5 is variable: some have a normal appearing optic cup and normal lens pit, others a distorted optic cup with a rudimentary lens, and the most affected have a persistent optic vesicle with no evidence of lens development (166). This phenotypic variation may reflect the action of modifier genes that interact with Gli3 during eye morphogenesis. The Gli3 xt-j allele, deleted for two-thirds of the 3 coding region of Gli3, is associated with poorly developed eyes, but the precise eye abnormalities have not been reported (165). Further investigation is required to deter-

10 1480 Human Molecular Genetics, 1996, Vol. 5, Review mine the specific eye cell types in which Gli3 is expressed, and to examine the role of Gli and Gli2 in eye development. Retinoic acid and retinoid receptors The retinoic acid and retinoid metabolites of vitamin A are critical for the proper development and maintenance of many structures, including the eye (review: 167). These metabolites are ligands of retinoic acid receptors (RARs) and retinoid receptors (RXRs), which are nonsteroid nuclear receptors that act as ligand-inducible transcription factors. The primary sequences of the RAR and RXR families are distantly related (168,169) and are characterized by the presence of two evolutionarily conserved domains, the ligand binding domain and the DNA binding domain (170,171) (Scheme 9). Scheme 9. Retinoic acid receptor and retinoid receptor proteins. The isoformspecific regions at the amino-termini are generated by differential splicing. The DNA-binding domain (DBD) and ligand binding domain (LBD) are indicated. Three genes encode retinoic acid receptors: RARα, RARβ, and RARγ (172), which map to human chromosomes 17q21, 3p24.3, and 12q13, respectively (see 145). The mouse homologues, Rarα, Rarβ, and Rarγ, map to mouse chromosomes 11, 14, and 15, respectively (see 6). The three retinoid receptor genes: RXRα, RXRβ, and RXRγ (173), map to human chromosomes 9q34, 6p21.3 and 1q22-q23, respectively (see 145), and the mouse homologues Rxrα, Rxrβ, and Rxrγ, map to mouse chromosomes 2, 17, and 1 (see 6). Multiple isoforms of the RARα, β, and γ and RXRβ and γ are generated by alternative promoter usage and alternative splicing, and are identified by a number, RARα1, 2; RARβ1, 2, 3, 4; RARγ1, 2; RXRβ1, 2 and RXRγ1, 2 (reviews: 168,169). The DNA binding domains of the RAR proteins are highly conserved with respect to each other, suggesting that the proteins can recognize similar DNA sequences (168). The RXR proteins are also conserved and recognize another set of related DNA target sequences. RARs and RXRs can form homodimers and heterodimers, adding complexity to retinoid dependent pathways (169,174). The combinatorial set of possible partners can explain the functional redundancy seen in gene targeting experiments (discussed below). Developmental expression of the Rar and Rxr genes. The Rar and Rxr genes are differentially expressed in the developing embryo (Rar expression reviews:172,175,176; Rxr expression review: 173). In the developing eye, Rarγ is not detected at all (175), but Rarα transcripts are expressed in all eye cell types (172). Rarβ is first expressed between E10.5 and E12.5 in the developing RPE, vitreous body and presumptive choroid. Rxrα and Rxrβ are both differentially expressed during early development, but specific eye expression data has not been reported (173). Rxrγ expression in the eye is detected in the neuroretina beginning at E13.5 (173). Mutations in the Rar and Rxr genes. Eye development is sensitive to disruption of retinoic acid levels, as seen by the varied defects which occur in mice born to vitamin A deficient mothers. Mutations of retinoic acid and retinoid receptors were therefore expected to affect eye development, and the effects of introducing null alleles of the Rarα, β, and γ, and Rxrα and β have been investigated (the Rxrγ gene has not yet been targeted) (177,178, review: 174). An Rxrα null mutation was lethal by E18.5, but before that age some mutants were still alive and could be examined (179). Although the optic cup appeared to be properly formed at E10.5, eyes examined between E12.5 and E16.5 showed ventrally restricted eye abnormalities, including a reduction of retinal size, abnormal corneal thickening (sometimes the cornea remains in contact with the lens), a reduced or absent anterior chamber, and coloboma of the optic nerve (179). Interestingly, mutation of the Drosophila Rxrα homologue, ultraspiracles (usp), yields a similar phenotype, in that the mutant flies have hypoplasia of the ventral retina (180). In contrast to Rxrα, the Rxrβ and Rarα, β and γ genes have been shown to be essential for eye development, but only when multiple null alleles are present in combination (177,178; reviewed in 174). The nine combinations of alleles that were found to lead to developmental eye abnormalities are: Rarα/Rarγ, Rarβ2/Rarγ, Rarα/Rarβ2, Rxrα +/ /Rarγ, Rxrα/Rarγ +/, Rxrα/Rarγ, Rxrα/Rarβ2 +/, Rxrα/Rarβ2, Rxrα/Rarα. (177,178, review: 174). As might be expected, these pairs of mutant alleles manifest many of the eye defects observed in vitamin A deficient animals, including: unfused or absent eyelids; absent or abnormal cornea, conjunctiva or anterior chamber; lens defects, including abnormal lens fibers, retrolenticular membrane, or ventral rotation; retinal defects, including ventral retinal reduction, retinal dysplasia, or coloboma; and coloboma of the optic nerve (174). In addition to phenotypic features which reflect abnormal morphogenesis, some null allele combinations cause abnormal differentiation, including the Rarα/Rarγ mutants in which the corneal epithelium keratinizes, and in Rarβ2/Rarγ mutants in which a fibrous retrolenticular membrane forms in the vitreous (177). Parenthetically, it should be noted that the phenotypic features that result from retinoic acid teratogenesis or mutations in the retinoic acid and retinoid receptors occur in patterns reminiscent of homeotic transformations (167,181). Several homeobox genes contain retinoic acid receptor elements in their regulatory regions, and their expression is directly responsive to retinoic acid levels, suggesting one mechanism for coordination of homeobox gene expression and patterning in embryogenesis (167,181). Other mammalian transcription factor genes that may be essential for eye development An additional 32 transcription factor genes have been reported to be expressed during mouse eye development (Table 2). Knowledge of the temporal and spatial expression pattern of these genes identifies the tissues and the stage of development at which their expression may be necessary. The requirement for most of these 32 genes in eye development is unclear, although it seems unlikely that all of them will prove to be required for eye formation or for the integrity of the mature organ. For most of these genes, no null allele mutants are yet available for analysis. In addition to the mutant genes listed in Table 1, null alleles of seven other genes have been created by gene-targeting. Published data documenting an absence of eye abnormalities is available only for two, Mash1 and Msx1. Mash1-deficient mice have no alterations in the size of the eye or in the gross organization of the retina (122), and homozygous null Msx1 mice appear to have normal ciliary bodies, a primary area of Msx1 expression (182).

11 1481 Human Nucleic Molecular Acids Genetics, Research, 1996, 1994, Vol. Vol. 5, 22, Review No No mention of the eye phenotype has been made in the description of homozygous mutants of five other genes: Isl1 (183), Dlx2 (184), Msx2 (28), Otx2 (185) and Nkx-2.1 (186). Table 2. Transcription factors expressed in the developing mammalian eye Presumably, these mice do not have gross eye abnormalities, but careful study must be made to determine whether eye function and structure are entirely normal.

12 1482 Human Molecular Genetics, 1996, Vol. 5, Review Table 2. continued Genes expressed earliest are listed first (as determined in mouse or rat). The increasingly shaded line denotes the progression of eye development: E8.5 is embryonic day 8.5, P0 dnotes post-natal day 0. A line that ends between P0 and Adult indicates that expression ends during post-natal development. Dots indicate that expression has not been determined, or not reported precisely. A dashed line indicates varied reports of expression. The transcription factor motif associated with each gene is indicated: homeobox (HB) and their accompanying class (prd, msh, POU, CVC, LIM, otd, dll, cut, NK); winged helix motif (WH); basic Helix-Loop-Helix domain [(b)hlh]; zinc-finger motif (ZnF); High Mobility Group protein (HMG) and steroid receptors. Genetic nomenclature is from the Mouse Genome Databse (6). Abbreviations: GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; RPE, retinal pigment epithelium. Accepted gene symbols: Hfhbf1,2 (HNF-3 forkhead, brain factor 1,2), Aporp1 (Apolipoprotein regulatory protein 1, was COUP-TFII/ARP-1), Erbal2 and 3 (avian erythroblastic leukemia viral (v-erb-a) oncogene homolog-like 2,3 (was EAR3/COUP-TFI), Nhlh1 (Nescient helix loop helix 1, was NSCL), Lhx2 (LIM homeobox protein 2, was LH-2), Nkx-2.1, 2.2 [NK1 transcription factor related, locus 2.1 (was TTF-1) and 2.2], Cutl1 (cut homeobox-like 1, was mclox) and Pou4f1,2,3 (POU class IV factor 1,2,3, was Brn-3a,-3b,-3c). Table 3. Evolutionary conservation of genes involved in eye development Drosophila melanogaster transcription factors required for eye development that have vertebrate homologues expressed in the eye. Papers cited are the original cloning papers or ones that show the relevance to eye development. Also indicated are human and rodent homologues with unknown or not required functions in eye development. Conservation of Drosophila eye development genes in vertebrates Several homologues of transcription factor genes required for the development of the Drosophila eye, such as Pax6, have also proved to be necessary for the mammalian eye (Table 3). Furthermore, many important mammalian transcription factor genes have been cloned by virtue of their homology to a critical Drosophila gene, an example being Hes1, a mouse homologue of hairy/ Enhancer of split (114). These genes, Pax6 and Hes1, are powerful evidence of the conservation of the mechanisms of eye development (43,113,123). Consequently, it will likely be fruitful to continue to study the mammalian homologues of other genes that are necessary for the formation of the invertebrate eye. Thus, some of the genes listed in Table 3, such as sine oculis (187) and seven-up (188), have been demonstrated to be essential for the Drosophila eye, but the importance of their homologues for mammals is unknown (189,190). Some vertebrate homologues from species other than