Journal of Neuro-Ophthalmology, December 2002, Volume 22, Issue 4

Developmental Neurogenetics and Neuro-Ophthalmology

The field of developmental neurogenetics has burgeoned over the past decade. Through the combined efforts of developmental biologists, geneticists, and clinicians, genetic defects resulting in neuro-ophthalmic disorders such as holoprosencephaly, microphthalmia, dominant optic atrophy, and optic nerve colobomas have been identified and characterized at the molecular level. Experimental studies in model organisms are continuing to identify novel genes critical for ocular and central nervous system development. Mutations in some of these genes have revealed a spectrum of pathology similar to that observed in septo-optic dysplasia, Möebius syndrome, and Duane retraction syndrome. This review examines our current knowledge of the molecular genetics of neuro-ophthalmic disease and focuses on several candidate genes for afferent and efferent visual system disorders.

NANOS SYMPOSIUM 2002 Developmental Neurogenetics and Neuro- Ophthalmology Jeffrey L. Bennett, MD, PhD The field of developmental neurogenetics has burgeoned over the past decade. Through the combined efforts of de­velopmental biologists, geneticists, and clinicians, genetic defects resulting in neuro- ophthalmic disorders such as ho-loprosencephaly, microphthalmia, dominant optic atrophy, and optic nerve colobomas have been identified and char­acterized at the molecular level. Experimental studies in model organisms are continuing to identify novel genes critical for ocular and central nervous system development. Mutations in some of these genes have revealed a spectrum of pathology similar to that observed in septo- optic dyspla­sia, Moebius syndrome, and Duane retraction syndrome. This review examines our current knowledge of the mo­lecular genetics of neuro- ophthalmic disease and focuses on several candidate genes for afferent and efferent visual system disorders. ( JNeuro- Ophthalmol 2002; 22: 286- 296) The development of the visual system occurs through the coordinated action of four major processes: the replica­tion of neuronal precursors, the determination of cellular identity, axonal pathfinding, and the establishment and maintenance of mature neurons and their connections. Neuro- ophthalmic disorders may be associated with abnor­malities in any one of these developmental processes. Ge­netic mutations disturbing early events, such as cell- type determination, usually result in significant developmental abnormalities across multiple organ systems, whereas mu­tations in later processes, such as neuronal maintenance, may lead to more isolated defects. In the following sections, I will review our current knowledge of anterior visual sys­tem and brainstem development and examine how muta­tions in key players in these pathways may result in neuro-ophthalmologic disorders. Departments of Neurology and Ophthalmology, University of Colora­do Health Sciences Center, Denver, Colorado. Address correspondence to Jeffrey L. Bennett, MD, PhD, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B- 182, Denver, CO 80262, USA; E- mail: jeffrey. bennett@ uchsc. edu Peer reviewed and modified from an oral presentation at the 28th An­nual Meeting of the North American Neuro- Ophthalmology Society, Cop­per Mountain, Colorado, February 9- 14, 2002. AFFERENT VISUAL SYSTEM Eye Development The development of the anterior visual system begins with the emergence of the optic vesicles from the lateral forebrain ( Fig. 1A). As development proceeds, the op­tic vesicle begins to extend toward the head ectoderm. Within it, retinal precursor cells ( RPCs) are established through the coordinated expression of a critical array of transcription factors: Rx, Pax6, Six3, Six6 ( OptxZ), Hesl, and Lhx2 ( 1- 5). Mutations in the Pax6, Six3, and Six6 genes are known to result in various abnormalities in hu­man eye and forebrain development including aniridia, holoprosencephaly, microphthalmia, and anophthalmia ( Table 1) ( 6- 9). When the optic vesicle makes contact with the over­lying head ectoderm, it induces the tissue to thicken and form the lens placode ( Fig. IB). After induction, the lens placode invaginates to form the lens pit, and the central portion of the underlying optic vesicle depresses to form the optic cup ( Fig. 1C). Soon afterwards, the lens pit pinches off to form the lens vesicle ( Fig. ID). Failure of lens induction is observed in mutations of the Pax6 ( 10), Lhx2,( l 1) and GH3 genes( 12) and may result from ab­normal function of either the optic vesicle or head ecto­derm. For instance, the Lhx2 transcription factor appears to be necessary for the production of a lens induction fac­tor by the optic vesicle ( 11). In contrast, Pax6 appears to act in the head ectoderm to initiate lens placode forma­tion ( 13). As development proceeds, the optic cup and optic stalk differentiate into the retina and optic nerve tract, re­spectively. The optic disc, the boundary zone between the optic cup and optic stalk, has been shown to play an impor­tant role in repressing retinal development by optic vesicle cells and directing closure of the optic fissure. Recent stud­ies have identified several factors important for the deter­mination and function of the optic disc: sonic hedgehog ( Shh), Pax2, and GU3. Deficient Shh expression in ze-brafish leads to cyclopia, whereas ectopic expression re­sults in small optic cups and enlarged optic stalks ( 14). Pax2 null mutant mice fail to form optic discs, resulting in medial extension of retinal pigment epithelial cells into the optic stalk, failure of axons to cross at the optic chiasm, and optic Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 286 J Neuro- Ophthalmol, Vol. 22, No. 4, 2002 NANOS SYMPOSIUM 2002 JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 Optic Vesicle Optic Vesicle \ Optic Stalk \ Lens Head Ectoderm g ode Optic Cup N Optic Cup Lens Pit FIG. 1. Morphogenic stages in ocular de­velopment. A: Emergence of the optic vesicle from the ventral forebrain. B: Induc­tion of the lens placode. C: Invagination of the optic cup and optic pit. D: Formation of the lens vesicle and closure of the em­bryonic fissure. Lens Vesicle nerve coloboma ( 15,16). The murine GH3 mutant, ' extra-toes,' also has optic nerve coloboma ( 12). Mutations in the human Shh and Pax2 genes are known to result in holo-prosencephaly and the renal- coloboma syndrome ( Table 1) ( 17,18). In the developing optic cup, retinogenesis proceeds in a fixed chronologic sequence. Retinal ganglion cells are generated first, followed in overlapping waves by horizon­tal cells, amacrine cells, photoreceptor cells, bipolar cells, and Muller glia cells ( 19). During retinogenesis, RPCs are maintained in an undifferentiated state by the action of Notch- Delta signaling. Under the combined influence of extrinsic cues and intrinsic signals, RPCs undergo a limited number of cell divisions and then differentiate into a re­stricted range of cell fates. The Pax6 gene is responsible for initiating vertebrate retinogenesis, and in the absence of Pax6 expression, RPC identity fails to be established. Dur­ing retinogenesis, continuedPord expression is required for the generation of all retinal cell types with the exception of amacrine cells ( 20). Downstream transcription factors such as Brn3b, NeuroD, Crx, and ChxlO are critical for the de­termination of retinal ganglion cells, amacrine cells, photo­receptor cells, and bipolar cells, respectively ( 21- 24). Hu­man developmental disorders resulting from mutations in the Crx and ChxlO genes have been identified ( Table 1) ( 25- 28). Genetic Disorders: Eye Development Due to multiple roles in organogenesis, severe defi­ciencies in proteins important for eye development usually result in significant developmental anomalies. The result­ing defects in eye and brain development are not subtle and are usually recognized quickly after birth. The ability of mutations such as amino acid substitutions or protein trun­cations to result in milder phenotypes remains to be deter­mined. A partial list of disorders resulting from deficiencies in genes important for eye development is provided in Table 1. A more detailed discussion of some of these disor­ders is presented below. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 287 JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 NANOS SYMPOSIUM 2002 TABLE 1. Developmental genes and neuro- ophthalmic disorders Vertebrate gene Gene expression pattern Human developmental disorder Developmental genes Pax6 Six3 Six5 Six6 ( Optx2) Shh Hesxl ChxlO Crx Hoxbl/ b2 Nkx6.1 Neural Pathfinding Pax2 Netrin- 1 DCC Ephrins Eph Receptors Robo Slit Cell growth TS NF1 Rb Cell maintenance OPA1 WFS1 XPD/ CSB Optic vesicle, lens placode, retina Lens placode, optic vesicle, hypothalamus, RPE Adult eye, lens Optic vesicle Ventral and midline brain Ventral diencephalon, pituitary Inner nuclear layer Retina Brainstem ( rhombomeres 4 & 5) Brainstem ( rhombomere 5) Optic stalk, optic chiasm Optic disc Retinal ganglion cell axons Brainstem Pathfinding axons Retina, optic chiasm, diencephalon Retina, optic chiasm, diencephalon Pan- expressed Pan- expressed Pan- expressed Pan- expressed Pan- expressed Pan- expressed Aniridia, anophthalmia, Peters anamoly6' 7' 31 Holoprosencephaly, microphthalmia8 Myotonic dystrophy98' 99 Anophthalmia, Septo- optic dysplasia ( C) 9 Holopro sencephaly: 7 Septo- optic dysplasia48 Microphthalmia2 8 Leber's congenital amaurosis, cone- rod dystrophy, retinitis pigmentosa25- 27 Mobius syndrome ( Q100' 101 Duane syndrome ( C) 87 Renal- coloboma syndrome16' 54 Septo- optic dysplasia ( C) 44 Optic nerve hypoplasia ( C), Septo- optic dysplasia ( C) 44 NR NR NR NR Tuberous sclerosis102 Neurofibromatosis103 Retinoblastoma Dominant optic atrophy67' 68 Diabetes mellitus with optic atrophy73 Cerebro- oculo- facio- skeletal syndrome75' 76 ( C), candidate locus; NR, None reported. Anophthalmia Absence of ocular structures ( anophthalmia) is the most dramatic phenotype resulting from a disturbance in eye development. Anophthalmia has been described as a consequence of deficiency of either the Pax6 or Six6 gene ( 7,9). Pax6 and Six6 are evolutionarily conserved proteins expressed during eye development in a variety of species ranging from Drosophila to vertebrates. Both Pax6 and Six6 are expressed in the earliest stages of eye development and are critical for determining the identity of RPCs. Pax6 is the master control gene governing eye mor­phogenesis in mammals, amphibians, fish, sea urchins, squid, nematodes, and planarians. Pax6, a member of the vertebrate paired box family of transcription factors, is highly conserved in evolution. Both the protein- coding and gene- regulatory regions of the Pax6 locus are conserved between flies and mice. Targeted expression of Pax6 genes from mouse, Drosophila, and Xenopus are capable of generating ectopic eyes in fly and frog embryos ( 29, 30), whereas mutations of the Pax6 locus in these or­ganisms severely disrupt ocular development. A natural mutation in the mouse Pax6 gene, Small eye ( Sey), is inher­ited in a semidominant fashion. Seyl+ heterozygous ani­mals exhibit abnormalities in the cornea and lens, whereas Sey I Sey homozygous animals are anophthalmic ( 10). The homozygous mutation is lethal to mouse embryos due to significant midline brain and facial defects. In humans, mutations in the Pax6 gene result in sig­nificant ocular abnormalities. Homozygous mutations re­sult in anophthalmia, nasal hypoplasia, and central nervous system defects. Haploinsufficiency of the Pax6 gene results in aniridia. In aniridia, the iris and foveal hypoplasia is often accompanied by corneal and lenticular abnormalities re­sulting in progressive glaucoma and optic atrophy. Muta­tions in the Pax6 gene are also associated with congenital central corneal opacity ( Peters anomaly) ( 31). Ocular Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 288 © 2002 Lippincott Williams & Wilkins NANOS SYMPOSIUM 2002 JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 abnormalities associated with Peters anomaly include glau­coma, microcornea, microphthalmia, colobomas, aniridia, and dysgenesis of the angle and iris. Six6 is a member of the . Six- type homeobox gene fam­ily conserved from Drosophila to man. The first Six family gene to be identified was Drosophila sine oculis ( so), a gene essential for compound eye formation. Since the initial characterization of the so gene, Six genes have been identi­fied in numerous species; and three members, Six3, Six5, and Six6, have proved critical for vertebrate eye and fore-brain development. Overexpression ofXenopus Six6 during development results in increased eye size, and ectopic expression of Six6 in differentiated retinal pigmented epi­thelium ( RPE) cells results in induction of downstream transcription factors critical for retinogenesis ( 32,33). Hap-loinsufficiency of human Six6 causes multiple abnormali­ties in the ventral forebrain and anterior visual system. Pa­tients are born with bilateral anophthalmia, the absence of optic nerves and chiasm, and pituitary abnormalities. Given these defects, it is possible that additional Six6 mutations may be responsible for some instances of septo- optic dys­plasia. Holoprosencephaly Holoprosencephaly ( HPE) is the most common de­velopmental defect of the forebrain in humans ( 34). HPE may result from either genetic abnormalities or teratogene-sis. Central nervous system ( CNS) abnormalities in HPE have been divided into three major categories: alobar HPE, semilobar HPE, and lobar HPE. Alobar HPE is character­ized by complete failure of forebrain division. In semilobar HPE, interhemispheric division occurs posteriorly, while in lobar HPE, there is separation of most of the cerebral hemi­spheres and ventricles. In general, the facial phenotype of HPE is proportional to the severity of the CNS changes. Minimal facial dysmorphism is usually seen in individuals with semilobar and lobar HPE, but occasionally facial and brain anomalies do not correlate. In humans, mutations in the ocular developmental genes Shh and Six3 result in HPE. Significant intrafamihal variability is noted among heterozygous Shh and Six3 pa­tients. Some individuals may be severely affected while others may appear phenotypically normal. This phenotypic variability may result from unidentified mutations in the same or additional genes, environmental factors, or back­ground gene expression. Six3 is a member of the previously described Six gene family. Six3 is expressed in the rostral neural plate, optic vesicle, and ventral forebrain. Ectopic expression of Six3 in zebrafish results in enlargement of the optic stalk and fore­brain ( 35). The human Six3 gene maps to chromosome 2p21, the HPE2 locus. Mutational analysis of the human Six3 gene in affected individuals has revealed multiple mu­tations in the homeodomain of the protein. These mutations are predicted to interfere with transcriptional activation ( 8). The Shh gene is critical for the development of mul­tiple vertebrate organ systems. As a result, targeted deletion of the Shh gene in mice results in forebrain defects, limb anomalies, and cyclopia ( 36). During eye development, Shh signaling plays a critical role in determining whether optic vesicle cells adopt a retinal or optic stalk fate ( 14). In vitro, Shh has been implicated in photoreceptor cell differentia­tion and survival ( 37). Human Shh maps to chromosome 7q36, the HPE3 locus. In HPE patients, multiple mutations have been identified in the Shh gene ( 38). Shh accounts for roughly 17% of familial and 3.7% of sporadic HPE. Microphthalmia Microphthalmia is a clinically heterogeneous devel­opmental disorder characterized by small eyes and other associated ocular abnormalities. In humans, multiple differ­ent microphthalmia loci have been identified. One is asso­ciated with the Six3 gene ( 8), while a second, on chromo­some 14q24.3, maps to the retinal homeobox gene ChxlO ( 28). ChxlO is a transcription factor important for retinal progenitor cell proliferation and bipolar cell differentiation ( 24). Autosomal recessive microphthalmia resulting from ChxlO mutation is associated with congenital cataracts and iris colobomas. The observed mutations in the ChxlO gene severely disrupt ChxlO DNA binding and transcriptional activation. Septo- optic Dysplasia Septo- optic dysplasia ( SOD), or de Morsier syn­drome, is a heterogeneous condition composed of optic nerve hypoplasia, absence of the septum pellucidum, and thinning or agenesis of the corpus callosum ( 39,40). Asso­ciated CNS malformations may include schizencephaly, cortical heterotopia, pituitary hypoplasia, encephalomala-cia, and periventricular leukomalacia. While most instances of SOD are sporadic, familial cases associated with autoso­mal recessive inheritance have been described ( 41,42). Tar­geted mutations in the murine homeobox gene Hesxl and the axon guidance molecules netrin- 1 and DCC ( deleted in colorectal cancer) result in phenotypes similar to patients with SOD ( 43,44). In mice missing Hesxl, abnormalities include absent or hypoplastic optic vesicles, pituitary abnormalities, reduction in prosencephalic tissue, and ab­normal morphogenesis of the corpus callosum and septum pellucidum ( 43). Hypothalamic abnormalities, optic nerve hypoplasia, and absence of the corpus callosum are ob­served in netrin- 1- and £> CC- deficient mice ( 45- 47). While genetic mutations in the human netrin- 1 and DCC genes have not been described, homozygous muta­tions in the Hesxl gene have been identified in two siblings Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 289 JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 NANOS SYMPOSIUM 2002 with optic nerve hypoplasia, absence of the corpus callo-sum, and hypoplasia of the pituitary gland ( 43). Five addi­tional mutations in Hesxl have recently been observed in children with sporadic pituitary disease and SOD ( 48). Mutations have clustered in the DNA- binding region of the protein consistent with a presumed loss in protein func­tion. Formal examination of homeobox genes with expres­sion patterns similar to Hesxl, such as Six3 and Six6, may yield additional genes responsible for both sporadic and fa­milial SOD. Neural Pathfinding The development of the afferent visual system re­quires the formation and maturation of neural pathways from the retina to targets in the CNS. Over recent years, studies by numerous investigators have begun to identify and characterize those molecules important for the guid­ance of retinal ganglion cells from the eye to targets in the thalamus and tectum. In mice, deficiencies in several pro­teins have been shown to result in abnormalities in RGC axon targeting, RGC axon trajectories, optic chiasm forma­tion, and axon guidance through the optic disc. Character­ization of these mutant animals has yielded insights into the mechanisms that govern the topographic organization of the visual fields and eventually may help identify candidate genes for neuro- ophthalmic disorders such as optic nerve colobomas and chiasmal agenesis. In the developing visual system, RGC axons are di­rected to their targets by both intrinsic and extrinsic cues. A major class of such cues is ephrins, membrane- bound ligands that signal through the Eph family of tyrosine ki­nase receptors. Ephrins routinely act as repellent cues to guide axon pathfinding during nervous system develop­ment ( 49). Targeted deletion of several Eph receptors and ephrins has produced a variety of axonal pathfinding de­fects in mice. These include abnormal forebrain axonal commissures, misdirected dorsal midbrain axonal connec­tions, and aberrant retinal axon guidance. Ephrin- A5 null mice demonstrate abnormal arborization of RGC axons in the superior colliculus ( 50). Mutations in human ephrins or Eph receptors, however, have yet to be described. Axonal guidance at the optic disc appears to require signaling between netrin- 1 and its receptor DCC ( 47). DCC, which is expressed on RGC axons, interacts with ne­trin- 1 in the developing optic disc to direct axonal growth into the optic stalk. In the absence of netrin- 1 ox DCC, RGC axons fail to exit into the optic nerve ( 44). The result is optic nerve hypoplasia and ectopic axonal growth within the retina. Additional abnormalities include hypothalamic changes and absence of the corpus callosum. As noted above, mutations in human netrin- 1 and DCC have yet to be described but may cause some instances of SOD. Formation of the optic chiasm requires the coordi­nated action of a variety of proteins critical for diencephalic development and axonal pathfinding. These include GAP- 43, Veal, Slit, Robo, and Pca2. In the absence of GAP- 43, RGC axons fail to progress through the chiasm and enter into the optic tract ( 51). The result is an enlarged and dis­organized optic chiasm. Targeted deletion of the Vaxl gene results in several defects in anterior visual system develop­ment including optic nerve colobomas, chiasmal agenesis, abnormalities in the anterior commissure and corpus callo­sum, and absence of optic nerve myelination ( 52). The axon guidance ligand Slit and its receptor Robo appear to de­termine the relative position of the optic chiasm at the ventral midline of the developing hypothalamus ( 53). Dos­ages of Slit and Robo may be important for determining whether a chiasm is normal, prefixed, or postfixed. RGC axons in Pax2 null mutant mice fail to cross at the optic chiasm and enter the ipsilateral optic tract. Additional ab­normalities in Pax2 mutant mice include optic nerve coloboma, midbrain defects, kidney hypoplasia, and inner ear malformations ( 16). Optic Nerve Coloboma Colobomas of the optic nerve result from incomplete or abnormal closure of the primitive embryonic fissure. In certain instances, the defect may extend to involve the cho­roid, retina, ciliary body, and iris. Mutations in the human Pax2 gene result in the renal- coloboma syndrome ( 54). The renal disease may vary significantly from patient to patient. Associated abnormalities include small dysplastic kidneys, vesico- ureteral reflux, and high frequency hearing loss. While the renal disease may be quite variable, all patients identified with mutations in the Pax2 gene have been ob­served to have colobomatous optic nerves. Iris colobomas have not been documented with Pax2 mutations. Cellular Proliferation Genes important for cell proliferation, " tumor sup­pressor" genes, are critical for governing the controlled rep­lication of cellular precursors during development. A num­ber of these " tumor suppressor" genes result in significant neuro- ophthalmic consequences, particularly those result­ing in phakomatoses or ocular tumors. A discussion of all of these disorders is well beyond the scope of this review, but some, due to their well described neurogenetics, de­serve mention. Tuberous Sclerosis Tuberous sclerosis ( TS) is an autosomal dominant syndrome that results in hamartomas in multiple organ sys­tems. Neurologic features generally predominate as a result of cortical tubers, subependymal nodules, and giant cell Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 290 © 2002 Lippincott Williams & Wilkins NANOS SYMPOSIUM 2002 JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 astrocytomas. Neuro- ophthalmic findings include retinal astrocytic hamartomas, papilledema, colobomas, and optic atrophy. TS results from loss- of- function mutations in one of two genes, TSC1 ( hamartin) or TSC2 ( tuberin) ( 55,56). Together, both mutations account for roughly 90% o of the disease spectrum. TS hamartomas generally follow a two- hit model of molecular pathogenesis. In this model, a germline mutation in an allele of TSC1 or TSC2 is accompanied by a second somatic mutation in the other allele ( 57,58). Mice deficient in TSC2 develop renal, lung, and liver tumors with a low rate of malignant conversion ( 59). The cellular function of hamartin and tuberin remain unknown, but early evidence suggests a role in GTPase signaling ( 60). Neurofibromatosis Type I The diagnostic criteria for neurofibromatosis Type 1 ( NF1) includes two or more of the following: cafe- au- lait skin lesions, cutaneous neurofibromas, optic gliomas, iris hamartomas ( Lisch nodules), axillary or inguinal freckling, sphenoid wing dysplasia, or a family history of NF1. The disorder arises from mutation of the NF1 gene product on chromosome 17. The NF1 protein contains a GTPase acti­vating domain ( GAP) and is presumed to function as a tu­mor suppressor gene by silencing the activated form of the T^^ proto- oncogene. Many of the documented NF1 muta­tions, however, lie outside the protein's GAP domain. Mutations are detected throughout the NF1 gene without the presence of a particular " hot spot". As a result, commercial mutational analysis is of limited value, and prenatal diagnosis is rarely useful in clinical practice. Mu­tational analyses have failed to identify a relationship be­tween genetic mutations and the severity of the NF1 phe-notype. Large deletions, however, appear to result in more severe abnormalities. As a true " tumor suppressor" gene, the molecular pathogenesis of NF1 is thought to depend on the somatic loss of NF1 heterozygosity or the generation of a second mutation in a related " tumor suppressor" gene. Documen­tation of such a mechanism has been fraught with technical difficulties due to the size of the NF1 gene and heteroge­neous composition of the tumors. Recent investigations have suggested that the Schwann cell is the primary cell type responsible for the generation of benign neurofibro­mas. Genetic analyses of this cell type in benign tumors have demonstrated loss of heterozygosity of NF1 at the mo­lecular level ( 61). Retinoblastoma Retinoblastoma is the most common malignant ocu­lar tumor of childhood ( 62). Approximately 30% o of retino­blastomas are bilateral and 70% o are unilateral. Roughly 40% are familial and 60% o are sporadic. The disorder generally presents by 15 months of age, and presentation after the age of 5 is rare ( 63). While most patients are iden­tified due to the presence of leukocoria, patients may present with strabismus, glaucoma, mydriasis, hyphema, or ocular inflammation. Retinoblastoma results from mutation of the Rb gene on chromosome 13ql4 ( 64). As mentioned previously for TS and NF1, loss of heterozygosity of the Rb gene must occur before a cell undergoes malignant transformation. Because of the high rate of spontaneous mutations in the Rb gene, 94% o of retinoblastoma cases are sporadic and the risk of genetic transmission ranges from 1% to 8%. Approxi­mately 50% to 90% of mutations in the Rb gene may be identified by current molecular genetic techniques. In cases where direct techniques fail, indirect analysis using restric­tion fragment length polymorphisms or microsatellite markers may prove useful. Given the somatic nature of the second mutation, tumor is the ideal tissue source for genetic analyses. In familial cases, alternative sources such as leu­kocytes may suffice. Cellular Metabolism and Maintenance Development and maturation of the vertebrate visual system requires the interaction of cell death and survival pathways. Abnormalities in cellular metabolism and main­tenance are common causes of cell loss through apoptosis or cell necrosis. For example, in the retina, errors in cellular metabolism may result in degenerative disorders such as Stargardt macular dystrophy, Leber congenital amaurosis, and cone- rod dystrophy. Analogously, in the optic nerve, energy depletion due to altered mitochondrial function has been hypothesized to cause disorders such as Leber heredi­tary optic neuropathy ( LHON) and dominant optic atrophy. Due to the wide array of genes involved in cellular main­tenance and metabolism, a complete review of such neu-rodevelopmental disorders is beyond the scope of this review. I will highlight three disorders with neuro-ophthalmic defects that have begun to be characterized at the molecular level. Autosomal Dominant Optic Atrophy Autosomal dominant optic atrophy ( ADOA; Kjer type) is the most frequent form of hereditary optic neurop­athy, with a prevalence of between 1: 12,000 and 1: 50,000. ADOA generally presents in the first decade of life and is clinically characterized by progressive loss of visual acuity, color vision disturbances, bilateral optic disc pallor, and paracentral and cecocentral scotomas ( 65). The clinical ex­pression of ADOA displays wide variation both within and between families demonstrating a vast range of visual acu­ities and color vision abnormalities. The disorder is geneti­cally heterogeneous, with two confirmed loci on chromo­somes 3q28- q29 ( OPA1) and 18ql2.2- ql2.3 ( OPA4) ( 66). Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 291 JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 NANOS SYMPOSIUM 2002 Recently, mutations in the OPA1 locus have been mapped to a dynamin- related GTPase protein ( 67,68). While most mutations appear to cluster in the GTPase do­main, the total spectrum of mutations is broadly distributed over the protein coding sequence ( 69,70). To date, most ob­servations support the notion that haploinsufficiency is the major pathogenic mechanism of ADOA. Unfortunately, there appears to be no tight genotype- phenotype correla­tion. Although the function of the OPA1 protein remains unknown, sequence similarities to the yeast Mspl and Mgml proteins suggest that OPA1 may play a role in main­taining mitochondrial integrity and function. No mutations in the OPA1 gene have been identified in LHON patients. Wolfram Syndrome Wolfram syndrome ( WS) is a progressive neurode­generative disorder characterized by juvenile- onset diabe­tes mellitus and progressive optic atrophy ( 71). WS is also known as DIDMOAD ( Diabetes Insipidus, Diabetes Melli­tus, Optic Atrophy, and Deafness). In addition to the hallmark optic atrophy and hearing loss, additional abnor­malities include ataxia, peripheral neuropathy, ptosis, psychiatric illness, and urinary tract atony. Because of the similarity of WS patients to individuals with deficiencies in oxidative phosphorylation, there is a postulate that WS re­sults from abnormalities in mitochondrial function. Genetic studies have linked WS to chromosome 4pl6. The gene WFS1 has recently been identified, and loss- of- function mutations have been characterized in mul­tiple independent kindreds ( 72,73). Detailed mutational analysis has revealed no genotype- phenotype relationships, mutational hot spots, or mutational clusters. Interestingly, no structural rearrangements or point mutations in mito­chondrial DNA ( mtDNA) have been identified in WSF1 pa­tients. Therefore, WS does not appear to be directly in­volved in mtDNA maintenance or repair. While portions of the WFS1 protein have homology to the alpha subunit of prenyl transferase, its cellular function remains uncertain. Cerebro- oculo- facio- skeletal Syndrome Cerebro- oculo- facio- skeletal Syndrome ( COFS) is a progressive disorder resulting in optic atrophy, brain atro­phy and calcification, cataracts, microcornea, joint contrac­tures, and growth retardation ( 74). The features of COFS are similar to Cockayne syndrome ( CS), a recessively in­herited neurodegenerative disorder caused by deficient cel­lular repair of UV- induced DNA damage. CS results from mutations in one of several DNA- repair genes: CSA, CSB, XPB, XPD, sadXPG. Recent investigations have identified cases of COFS associated with mutations in the XPG, XPD, and CSB genes ( 75,76). It is hypothesized that COFS represents a subtype of CS and results from impaired nucleotide excision repair of UV- damaged DNA. It is rec­ommended that tissue from affected or at risk individuals be screened for UV sensitivity or abnormalities in DNA repair. EFFERENT VISUAL SYSTEM Brainstem Development The hindbrain, or rhombencephalon, is composed of the cerebellum, pons, and medulla. With the exception of the oculomotor nuclei, each cranial nerve nucleus is derived from rhombencephalic neuronal precursors. Over the past several years, significant steps have been taken to charac­terize the molecular mechanisms that govern hindbrain development. The results of these investigations have yielded insights into the potential relationship between human developmental disorders and the molecular signals that determine neuronal identity and axonal pathfinding in the brainstem. In early development, the hindbrain is segmented into five compartments termed rhombomeres ( Fig. 2). Neuronal populations within individual rhombomeres display limited intermixing with neighboring compartments. As a result, the position of a neural progenitor during hindbrain seg­mentation determines its contribution to adult brainstem structure and axonal connections. For example, the trochle­ar, trigeminal, abducens, and facial motor neurons are gen­erated in rhombomeres 1, 2- 3, 5- 6, and 4- 5, respectively. Due to their compartmental identity, these neural progeni­tors display programmed migratory behaviors and project axons along defined trajectories toward their peripheral tar­gets ( Fig. 2). While a neural progenitor's position along the anteroposterior axis determines its identity, the cell's posi­tion along the dorsoventral axis appears to influence its classification as sensory or motor. Sonic hedgehog ( Shh) appears to be a key player in the process of neuronal class determination along the dorsoventral axis. Graded expres­sion of Shh along this axis appears to generate domains con­ducive to either motor ( ventral) or sensory ( dorsal) neuron development ( 77). The molecular mechanisms by which positional in­formation in the developing brainstem is translated into neuronal identity and axonal connections are currently un­der active investigation. Using " knockout" mice generated by gene- specific homologous recombination, families of homeobox transcription factors ( Hox, Kreisler, Nla, Phox, Krox20) and tyrosine kinase receptors ( Eph) have been shown to play important roles in rhombomeric compart-mentalization, neuronal precursor determination, and the establishment of specific brainstem axonal pathways. Among these gene families, several proteins stand out as candidate genes for developmental neuro- ophthalmic dis­orders such as Mobius syndrome, Duane retraction syn­drome, congenital third nerve palsies, and the Marcus Gunn jaw- winking phenomenon. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 292 © 2002 Lippincott Williams & Wilkins NANOS SYMPOSIUM 2002 JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 I l 3 5 S 5 " 5 a n. ^ % 3 I I High expression Low Expression Midbrain FIG. 2. Segmental expression of genes in­volved in patterning the brainstem. The re­lationship of the cranial nerve nuclei to rhombomeric segments is illustrated sche­matically on the right. The level of develop­mental gene expression in various rhom-bomeres is depicted to the left. Adapted with permission from Kandel ER, Schwartz JH, Jessell TM. Principles of Neural Science, 4th edn. New York: McGraw- Hill, 2000. Mobius Syndrome Mobius syndrome is characterized by facial diplegia and sometimes abducens palsy ( 82%), total external oph­thalmoplegia ( 25%), oculomotor palsy ( 21%), or ptosis ( 10%) ( 78). Less frequently there is involvement of the lower cranial nerves. Mobius syndrome is usually sporadic, and pathology varies from hypoplasia or aplasia of cranial nerve nuclei to more widespread brainstem destruction ( 79,80). Because some cases have widespread pathology, Mobius syndrome has been hypothesized to result from brainstem vascular insufficiency. The Hox transcription factors are expressed early in development and control critical steps inhindbrain segmen­tation and neuronal determination ( 81,82). The Hox genes are expressed in overlapping domains along the developing hindbrain and spinal cord ( Fig. 2). Cumulative data demon­strate that the Hox genes are responsible for conferring rhombomeric identity, determining neuronal cell fate, and directing axonal projections. For example, in Hoxal- mill mice, rhombomeres 3 through 6 fail to form, while in Hoxa2- nvl\ mutants, the rhombomere 1/ 2 boundary is miss­ing, the sizes of rhombomeres 2 and 3 are reduced, and tri­geminal motor neuron axons display abnormal projections. Hoxbl- and Hoxb2- nvl\ mutant mice display a facial diplegia that clinically resembles Mobius syndrome ( 83,84). In the absence of Hoxbl or Hoxb 2, there is failure to generate the facial motor nuclei, while the facial sensory nuclei remain intact. Hoxbl- and Hoxb2- xm\\ mice, however, fail to reproduce the additional brainstem abnor­malities commonly observed in Mobius syndrome. In Hoxbl- andHoxb2- nvl\ mice, cranial nerves III, IV, and VI, which develop outside of rhombomere 4, remain unaf­fected. Therefore, Mobius syndrome may arise from com­bined Hox gene mutations or defects in an additional brain­stem transcription factor important for Hox gene expres­sion. Additional candidate genes include Kreisler, Nkx6.1 ( see below), and Hoxa2. In combined Hoxa2- and Hoxbl-null mice, facial diplegia is accompanied by significant lower cranial nerve abnormalities ( 85). Mutations in either Nkx6.1 or the transcription factor Kreisler result in the loss of rhombomeres 5 and 6 and the absence of the abducens motorneurons ( 86,87). Duane Retraction Syndrome Duane retraction syndrome is characterized by co-contraction of the ipsilateral medial and lateral recti muscles ( 88). Three types of Duane retraction syndrome have been identified based on electromyographic record­ings: Type I) impaired abduction with normal adduction; Type II) impaired adduction with normal abduction; and Type III) impaired abduction and adduction ( 89). Associ­ated findings in Duane retraction syndrome include Marcus Gunn jaw- winking, gustatory- lacrimal synkinesis, sensori­neural deafness, Klippel- Feil spinal anomaly, cervico-oculo- acoustic syndrome, and thenar hypoplasia. Most cases of Duane syndrome are sporadic; rare familial cases Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 293 JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 NANOS SYMPOSIUM 2002 have been described. Pathology demonstrates absence of the abducens nerve and aberrant innervation of the lateral rectus by branches of the inferior division of the oculomotor nerve ( 90). A potential candidate gene for Duane retraction syn­drome is Nkx6.1. The Nkx family of transcription factors is required for the specification of populations of ventral mo-torneurons and interneurons in the developing hindbrain and spinal cord. In the absence of Nkx6.1, the rhombomere 5- specific abducens neurons are missing. Additional abnor­malities include absence of the hypoglossal nuclei and overabundance of Y1 neurons. Unfortunately, no examina­tion of oculomotor axon projections has been performed in mutant animals. Interestingly, Nkx expression patterns are altered in Hox mutants ( 91,92). This interrelationship be­tween Hox and Nkx gene expression may explain the skel­etal and brainstem abnormalities observed in some cases of Duane retraction syndrome and the frequent occurrence of abducens palsies in Mobius syndrome. Congenital Third Nerve Palsy Some congenital third nerve lesions may result from brain stem abnormalities. In some instances, autopsy stud­ies have revealed aplasia of the oculomotor nuclei or mal­formation of the mesencephalon ( 93). Phox2a is a ho-meobox transcription factor that is expressed in the locus ceruleus, sympathetic and parasympathetic ganglia, bran-chiomotor and visceromotor brainstem neurons, and the oculomotor and trochlear nuclei ( 94,95). Phox2a may rep­resent a candidate gene for congenital nuclear third nerve lesions. Given the widespread pathology evident in Phox2a- mA\ mice ( 96), mutations in Phox2a producing limited third nerve pathology would need to result in only partial loss- of- function in the Phox2a protein. Congenital Brainstem Synkineses Synkineses are frequently observed after congenital or acquired injury to the brainstem or cranial nerves. While synkinesis after acquired injury results from aberrant rein-nervation of peripheral targets by misguided regenerating axons, congenital synkineses, such as Marcus Gunn jaw-winking and gustatory- lacrimal synkinesis, may represent a developmental defect in axonal pathfinding. In Hoxa2- null mice, trigeminal motorneurons display abnormal axonal projections and target selection in the second branchial arch. Alterations in Hoxa2 gene expressions have been shown to directly abolish Eph- receptor expression in rhom-bomeres 2 and 4. EphA3 may be a candidate gene important for trigem­inal motor axon pathfinding. In the chick, EphA3 is ex­pressed on trigeminal motorneurons during their pro­jections to muscle targets, and corresponding ephrin- A ligands are produced by first branchial arch targets ( 97). Elucidation of the relationship between Eph receptors and brainstem axonal pathways may eventually help to decipher the molecular pathogenesis of congenital synki-netic abnormalities. CONCLUSIONS Development of the vertebrate visual system is a complex process demanding the coordinated action of mul­tiple cellular genes. Over the past decade, considerable progress has been made in our understanding of the molecu­lar mechanisms that govern neurodevelopment. With that understanding, our knowledge of the genetic defects that cause neuro- ophthalmic disease has expanded greatly. The upcoming years offer great promise for the further molecu­lar characterization of neurogenetic disorders of the visual system. Ongoing research in developmental neurobiology, embryology, and clinical genetics will allow identification and further characterization of the mechanisms governing the growth, development, maturation, and maintenance of the visual system. REFERENCES 1. Walther C, Grass P. 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