Pax genes in development and maturation of the vertebrate visual system: implications for optic nerve regeneration

Pax genes play a pivotal role in development of the vertebrate visual system. Pax6 is the master control gene for eye development: ectopic expression of Pax6 in Xenopus laevis and Drosphila melanogaster leads to the formation of differentiated eyes on the legs or wings. Pax6 is involved in formation of ganglion cells of the retina, as well as cells of the lens, iris and cornea. In addition Pax6 may play a role in axon guidance in the visual system. Pax2 regulates differentiation of the optic disk through which retinal ganglion cell axons exit the eye. Furthermore, Pax2 plays a critical role in development of the optic chiasm and in the guidance of axons along the contralateral or ipsilateral tracts of the optic nerve to visual targets in the brain. During development Pax7 is expressed in neuronal cells of one of the major visual targets in the brain, the optic tectum/superior colliculus. Neurons expressing Pax7 migrate towards the pia and concentrate in the stratum griseum superficiale (SGFS), the target site for retinal axons. Together, expression of Pax2, 6 and 7 may guide axons during formation of functional retinotectal/collicular projections. Highly regulated Pax gene expression is also observed in mature animals. Moreover, evidence suggests that Pax genes are important for regeneration of the visual system. We are currently investigating Pax gene expression in species that display a range of outcomes of optic nerve regeneration. We predict that such information will provide valuable insights for the induction of successful regeneration of the optic nerve and of other regions of the central nervous system in mammals including man.     

Fiber optic mapping of the Xenopus visual system: shift in the retinotectal projection during development

Two new techniques for assaying the retina to tectum connections in the lower vertebrate visual system are presented. These techniques allow defined regions of the retina to be stimulated, thus circumventing some of the difficulties of the more conventional retinotectal mapping techniques. Applying these techniques to the Xenopus visual system demonstrates that the retina-to-tectum projection shifts during development. The central part of the retinotectal projection moves medially and caudally about 150 microns (10% of the size of the tectum) in two weeks. The presence of such plasticity in a normal developing animal indicates that the plasticity previously observed in experimentally altered animals probably reflects a normal developmental process.     

Embryonic retinal ablation and post-metamorphic optic nerve crush: effects upon the pattern of regenerated retinotectal connections.

We examined relationships between healing observed during embryonic Xenopus retinal and optic nerve regeneration and resultant visuotectal pattern formation. Dorsal (D) and nasoventral (NV) 1/3 sized eye fragments were surgically created in stage 32 Xenopus laevis embryos. Gross anatomical healing modes of these fragments were examined 2 days post-surgery (stage 43). Healing was categorized according to the degree of cell movements observed. Animals were reared through metamorphosis and electrophysiologic mapping techniques were employed on those animals whose eyes regenerated. All D 1/3 fragments showed normal (non-duplicated) projections to the tectum; most (80%) of the healing observed showed little cell movements (the remaining 20% showed substantial cell movements, yet failed to show duplicated projections). Most NV 1/3 fragments (73%) formed two mirror image projections to the contralateral midbrain optic tectum (pattern duplication). Most (88%) of the healing observed among these animals showed massive cell movements in the ventral retinal region (the remaining 12% showed moderate cell movements). The remaining NV 1/3 fragments (27%) showed moderate cell displacement and failed to show duplicated projections). These data are compatible with a cell-movement:intercalary cell division hypothesis in which duplication is dependent upon specific positional confrontation and subsequent cell division. In additional studies, in adult animals, the optic nerves of eyes with duplicated projections were crushed and allowed to regenerate for 1 year. Duplicated projections were restored, indicating that developmental and maturational factors are probably not responsible for duplicative pattern formation; rather, information intrinsic to the eye, possibly created during healing interactions and/or fiber ingrowth to the tectum, underlies duplicate innervation of the tectum.     

Dynamic aspects of retinotectal map formation revealed by a vital-dye fiber-tracing technique

In the visual system of Xenopus laevis, the axons from the retinal ganglion cells of the eye form a topographic projection onto the optic tectum. Many studies have focused on revealing the mechanisms responsible for this precise and regular projection pattern. In contrast to the static view of the system that one might expect from examining the regularity of the projection, recent work on its regeneration and its changes during larval development indicate that part of the patterning process involves the dynamic behavior of optic fibers. Typically, anatomical and electrophysiological techniques have been used to obtain static views of the developing retinotectal projection which then must be complied to provide a glimpse of any dynamic behavior. Here we report on experiments using a newly developed fiber tracing technique to directly follow the emergence of topography in the developing retinotectal projection. Defined halves of the developing eyebud were labeled with a vital fluorescent dye which fills the growing axons, and the projection of the labeled cells was followed for up to 2 weeks in individual animals. The experiments confirm that dorsal and ventral optic nerve fibers sort out into an ordered projection early in development. In contrast, nasal and temporal fibers initially overlap, and the same sets of prelabeled fibers then sort out into the adult topography over a period of days.     

The role of visual experience in the formation of binocular projections in frogs

Many parts of the visual system contain topographic maps of the visual field. In such structures, the binocular portion of the visual field is generally represented by overlapping, matching projections relayed from the two eyes. One of the developmental factors which helps to bring the maps from the two eyes into register is visual input. The role of visual input is especially dramatic in the frog, Xenopus laevis. In tadpoles of this species, the eyes initially face laterally and have essentially no binocular overlap. At metamorphosis, the eyes begin to move rostrodorsally; eventually, their visual fields have a 170 degree region of binocular overlap. Despite this major change in binocular overlap, the maps from the ipsilateral and contralateral eyes to the optic tectum normally remain in register throughout development. This coordination of the two projections is disrupted by visual deprivation. In dark-reared Xenopus, the contralateral projection is nearly normal but the ipsilateral map is highly disorganized. The impact of visual input on the ipsilateral map also is shown by the effect of early rotation of one eye. Examination of the tectal lobe contralateral to the rotated eye reveals that both the contralateral and the ipsilateral maps to that tectum are rotated, even though the ipsilateral map originates from the normal eye. Thus, the ipsilateral map has changed orientation to remain in register with the contralateral map. Similarly, the two maps on the other tectal lobe are in register; in this case, both projections are normally oriented even though the ipsilateral map is from the rotated eye. The discovery that the ipsilateral eye's map reaches the tectum indirectly, via a relay in the nucleus isthmi, has made it possible to study the anatomical changes underlying visually dependent plasticity. Retrograde and anterograde tracing with horseradish peroxidase have shown that eye rotation causes isthmotectal axons to follow abnormal trajectories. An axon's route first goes toward the tectal site where it normally would arborize but then changes direction to reach a new tectal site. Such rearrangements bring the isthmotectal axons into proximity with retinotectal axons which have the same receptive fields. Anterograde horseradish peroxidase filling has also been used to study the trajectories and arborizations of developing isthmotectal axons. The results show that the axons enter the tectum before the onset of eye migration but do not begin to branch profusely until eye movement begins to create a zone of binocular space.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alterations in the Xenopus retinotectal projection by antibodies to Xenopus N-CAM

The patterned neural projection from the eye to the optic tectum of lower vertebrates (the retinotectal projection) has been proposed to be ordered by interactions between the optic nerve fibers and their surrounding tissues. To investigate the role of one such defined cell interaction, agarose implants containing antibodies to the neural cell adhesion molecule, N-CAM, were inserted into the tectum of the African clawed frog, Xenopus laevis. Both monoclonal and polyclonal antibodies against N-CAM reversibly and specifically distorted the pattern of the retinotectal projection, decreasing the precision of the projection as determined by electrophysiological techniques as well as decreasing the density of retinal innervation of the tectum and the branching of single axons as determined by horseradish peroxidase tracing. The anatomical effects became maximal at 4 to 6 days after implantation and returned to undetectable levels by 2 weeks, whereas the physiological effects became maximal by 8 to 10 days and a normal physiological map was reestablished within 4 weeks. The results are consistent with the hypothesis that anti-N-CAM antibodies perturb the ongoing growth and retraction of the terminal arbors of the optic nerve fibers, such that a region of the tectum becomes largely denuded of fibers. The physiological defects may then be a consequence both of the initial retraction of optic nerve terminals and of the rapid ingrowth of the perturbed and neighboring optic nerve fibers into the denuded region after the antibodies were cleared from the tectum. These results support the concept of a major role for N-CAM-mediated adhesion during map regeneration and maintenance.     

Binocular maps in Xenopus tectum: Visual experience and the development of isthmotectal topography

Xenopus frogs have a prominent binocular field that develops as a consequence of the migration of the eyes during the remodeling of the head during and after metamorphosis. In the optic tectum, a topographic representation of the ipsilateral eye develops during this same period. It is relayed indirectly, via the nucleus isthmi. In the early stages of binocular development, the topographic matching of the ipsilateral input to the retinotectal input from the contralateral eye is largely governed by chemical cues, but the ultimate determinant of the ipsilateral map is binocular visual input. Visual input is such a dominant factor that abnormal visual input resulting from unilateral eye rotation can induce isthmotectal axons to alter their trajectories dramatically, even shifting their terminal zones from one pole of the tectum to the other. This plasticity normally is high only during a 3-4-month critical period of late tadpole-early juvenile life, but the critical period can be extended indefinitely by dark-rearing. N-methyl-D-aspartate (NMDA) receptors are involved in this process; plasticity can be blocked or promoted by chronic treatment with NMDA antagonists or agonists, respectively. Cholinergic nicotinic receptors on retinotectal axons are likely to play an essential role as well. Modifications in the polysialylation of neural cell adhesion molecule are correlated with the state of plasticity. The circuitry underlying binocular plasticity is not yet fully understood but has proved not to be a simple convergence of ipsilateral and contralateral inputs onto the same targets.     

Expression patterns of Ephs and ephrins throughout retinotectal development in Xenopus laevis

The Eph family of receptor tyrosine kinases and their ligands the ephrins play an essential role in the targeting of retinal ganglion cell axons to topographically correct locations in the optic tectum during visual system development. The African claw-toed frog Xenopus laevis is a popular animal model for the study of retinotectal development because of its amenability to live imaging and electrophysiology. Its visual system undergoes protracted growth continuing beyond metamorphosis, yet little is known about ephrin and Eph expression patterns beyond stage 39 when retinal axons first arrive in the tectum. We used alkaline phosphatase fusion proteins of EphA3, ephrin-A5, EphB2, and ephrin-B1 as affinity probes to reveal the expression patterns of ephrin-As, EphAs, ephrin-Bs, and EphBs, respectively. Analysis of brains from stage 40 to adult frog revealed that ephrins and Eph receptors are expressed throughout development. As observed in other species, staining for ephrin-As displayed a high caudal to low rostral expression pattern across the tectum, roughly complementary to the expression of EphAs. In contrast with the prevailing model, EphBs were found to be expressed in the tectum in a high dorsal to low ventral gradient in young animals. In animals with induced binocular tectal innervation, ocular dominance bands of alternating input from the two eyes formed in the tectum; however, ephrin-A and EphA expression patterns were unmodulated and similar to those in normal frogs, confirming that the segregation of axons into eye-specific stripes is not the consequence of a respecification of molecular guidance cues in the tectum.     

Retina development in zebrafish requires the heparan sulfate proteoglycan agrin

Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.     

Retrovirus-mediated gene expression during chick visual system development

The chick embryo is an excellent model for studying eye morphogenesis, retinal cell fate determination, and retinotectal projections due to its accessibility and the available molecular tools. Avian replication-competent retroviruses allow efficient infection of proliferating cells and stable integration of the viral genome, including up to 2.3kb of foreign cDNA, into the host chromosome. High-titer retroviruses are produced by transient transfection of avian DF-1 cells followed by centrifugation of the culture medium. Targeted infection of the optic vesicle, the lens vesicle, the retina and pigmented epithelium, the periocular mesenchyme, and the tectum can be performed at different developmental stages in ovo. In addition, retroviruses can be used to transduce genes of interest into various ocular tissue explants or cells in vitro. Virus-mediated gene expression can be detected within 12h of infection. Therefore, avian replication-competent retroviruses serve as powerful tools to misexpress wild-type and mutant gene products and to study molecular mechanisms underlying vertebrate visual system development.     

Optical properties of in situ eye lenses measured with X-ray Talbot interferometry: a novel measure of growth processes

The lens, a major optical component of the eye, has a gradient refractive index, which is required to provide sufficient refractive power and image quality. The refractive index variations across the lens are dependent on the distributions and concentrations of the varying protein classes. In this study, we present the first measurements of the refractive index in the in situ eye lens from five species using a specially constructed X-ray Talbot grating interferometer. The measurements have been conducted in two planes: the one containing the optic axis (the sagittal plane) and the plane orthogonal to this (the equatorial plane). The results show previously undetected discontinuities and fluctuations in the refractive index profile that vary in different species. These may be linked to growth processes and may be the first optical evidence of discrete developmental stages.     

Visual optics in toads (Bufo americanus)

Aspects of visual optics were investigated in the American toad (Bufo americanus). The development of the refractive state of the eye during metamorphosis was followed with IR photoretinoscopy. Frozen sections documented the changes in optical parameters before and after metamorphosis. There is a difference in light sensitivity between juvenile and adult toads. Binocular accommodation in adult toads was observed. 1. IR photoretinoscopic measurements showed that the refractive state of the eye changed very rapidly during metamorphosis, about 10 D/h while the animal entered the terrestrial habitat. 2. Frozen sections showed that the almost spherical lens in a tadpole eye had flattened in a just metamorphosed toad's eye while at the same time the distance of the lens to the retina had decreased. However, the morphological measurements were not sufficiently sensitive to record the relatively small changes in ocular dimensions that were responsible for the rapid changes in refractive state during metamorphosis. 3. Schematic eyes, with homogeneous and non homogeneous lenses, were constructed for tadpoles, juvenile toads, and adult toads. 4. Nonparaxial raytracing studies in schematic eyes suggested that the lenses of animals of the three developmental stages tadpole, juvenile toad, and adult are not homogeneous but have a refractive index gradient. The raytracing studies indicated that the refractive index gradient is different for the different developmental stages, being highest in the tadpole lens. 5. The observations of toads during feeding behavior at different light levels showed an increased light sensitivity in the adult nocturnal toads in contrast to the juvenile animals, which are diurnal. The increased light sensitivity could partly be explained with an increase in aperture and an increase in red rod outer segments. To fully explain the higher light sensitivity in adult toads, changes in neuronal parameters had to be assumed. 6. Retinoscopic measurements of the resting refractive state in the adult toad showed a hyperopic defocus of about +8 D. By subtracting the measurement artefact for retinoscopy, the true resting focus was found to be nearly emmetropic. 7. The amount of natural accommodation in adult toads during normal feeding behavior was investigated with IR photoretinoscopy. Binocular accommodation of about 8 D was observed.     

Expression of the forkhead transcription factor FoxN4 in progenitor cells in the developing Xenopus laevis retina and brain

Forkhead proteins are involved in gene regulation in a large variety of developmental situations. Several forkhead gene products are expressed in the developing eye and brain. Here we characterize the expression of FoxN4 during Xenopus development. We report that FoxN4 is expressed in the eye from the earliest stages of specification through retinal maturation. FoxN4 is also expressed in the pallium, optic tectum, isthmus, reticular formation, and in cells lining the ventricle of the tadpole brain.     

Specificity and retinotectal projections of quarter-eye fragments in Xenopus laevis

Three quarters of the eye anlage in Xenopus embryos of stage 33/34 were eliminated in three different sets of experiments. The remaining quadrant originated from the nasoventral part of the retina, from its ventral portion, or from the temporo-ventral area of the retina. All the fragments developed into small eyes of normal shape. The retinotectal connections did not deviate from those found in the control groups, even though mirror-image duplication was fairly frequent. For all fragments the tectal projection fields were rather limited. There was some indication of fragments retaining their original specificity. Irrespective, however, of their different origins, the optic projections always occupied the rostrolateral area of the tectum.     

Control of the development of the ipsilateral retinothalamic projection in Xenopus laevis by thyroxine: results and speculation

The ipsilateral retinothalamic projection of the frog Xenopus laevis is formed by the axons of a subset of retinal ganglion cells which are found throughout peripheral and non-nasodorsal retina. Unlike the crossed retinotectal and retinothalamic projections, which begin to form during early embryonic stages, the ipsilateral projection does not begin to develop until late in tadpole life, at stages when thyroxine first becomes detectable in the circulation. Blocking the production of thyroid hormone in tadpoles prevents the development of the ipsilateral projection, in a reversible manner. Intraocular injection of thyroxine can "rescue" the development of the projection in tadpoles which otherwise remain premetamorphic. In addition, the projection from one eye of a metamorphically-blocked tadpole can be induced to form by an intraocular injection of thyroxine at a dose which has no detectable effect on retinal development in the other, untreated eye. These results indicate that the development of the ipsilateral retinothalamic projection is dependent upon thyroxine, and strongly suggest that the hormone acts at the level of the eye, rather than at the optic chiasm or thalamic target, to bring about the development of a new pathway. A number of ways in which thyroxine might act in the system are discussed.     

Homotopic and heterotopic transplantations of quail tectal primordia in chick embryos: Organization of the retinotectal projections in the chimeric embryos

To study the adaptative capabilities of the retinotectal system in birds, the primordium of one optic tectum from 12-somite embryos of Japanese quail was transplanted either homotopically, to replace the ablated same primordium, or heterotopically, to replace the ablated dorsal diencephalon in White Leghorn chick embryos of the same stage. The quail nucleolar marker was used to recognize the transplants. The cytoarchitecture of the tecta and the retinal projections from the eye contralateral to the graft were studied on the 17th or 18th day of incubation in the chimeric embryos by autoradiographic or horseradish peroxidase tracing methods. Morphometric analysis was applied to evaluate the percentage of the tectal surface receiving optic projections. It was observed that: (i) quail mesencephalic alar plate can develop a fully laminated optic tectum even when transplanted heterotopically; (ii) retinal ganglion cells from the chick not only recognize the tectal neurons of the quail as their specific targets in homotopic grafts, but the optic fibers deviate to innervate the heterotopically grafted tectum; (iii) in the presence of a graft, the chick retina is unable to innervate a tectal surface of similar or larger size than that of the control tectum; (iv) tectal regions devoid of optic projections, whether formed by donor or by host cells, always present an atrophic lamination; (v) the diencephalic supernumerary optic tectum competes with and prevails over the host tectum as a target for optic fiber terminals.     

Early tissue interactions leading to embryonic lens formation in Xenopus laevis

Our previous research has demonstrated that lens induction in Xenopus laevis requires inductive interactions prior to contact with the optic vesicle, which classically had been thought to be the major lens inductor. The importance of these early interactions has been verified by demonstrating that lens ectoderm is specified by the time it comes into contact with the optic vesicle. It has been argued that the tissues which underlie the presumptive lens ectoderm during gastrulation and neurulation, dorsolateral endoderm and mesoderm, are the primary early inductors. We show here, however, that these tissues alone cannot elicit lens formation in Xenopus ectoderm. Evidence is presented that presumptive anterior neural plate tissue (which includes the early eye rudiment) is an essential early lens inductor in Xenopus. The presence of dorsolateral mesoderm appears to enhance this response. These findings support a model in which an essential inductive signal passes through the plane of ectoderm during gastrula and early neurula stages from presumptive anterior neural tissue to the presumptive lens ectoderm. Since there is evidence for such interactions within a tissue layer in mesodermal and neural induction as well, this may be a general feature of the initial stages of determination of many tissues.     

非洲爪蟾眼的形态发生研究

详细观察和描述了非洲爪蟾Xenopus laevis眼的发生和发育变化过程,并分别对各发育时期视网膜的厚度进行了定量分析.非洲爪蟾眼的发牛开始于眼原基的形成,进而形成视泡;晶状体的发生是在视杯外壁增厚的同时诱导覆盖其上的胚胎外胚层内层增厚,形成预定晶状体板;在视网膜和晶状体共同诱导下,预定角膜上皮变为透明的角膜.在视杯出现之前,预定RPE的厚度由厚变薄,NR层不断地增厚直至结构功能完善.     

Cross-species axial signaling, with realignment of retinal axes, in embryonic Xenopus eyes

The locus specificities which enable retinal ganglion cells to assemble a topographic retinotectal map are patterned about a pair of (anteroposterior and dorsoventral) retinal axes set down in the early eye bud. We have transplanted a Xenopus laevis eye bud, at stage $\text{23}\text{24}$ when the retinal field is still responsive to the axial signals from the surrounding tissues, into the enucleated eye socket of a comparable stage Ambystoma maculatum embryo. Three days later, when the Xenopus eye had reached early larval stages and was no longer responsive to extraocular signals, the eye was retransplanted into the socket of the Xenopus final carrier embryo. The pattern of retinotectal connections between the eye and the carrier's optic tectum was examined by electrophysiological analysis of the visuotectal projections. The results indicated that many of the retinae had patterned locus specificities about axes derived from the salamander intermediate host. We infer that axial signaling involves fundamental cellular processes which have been highly conserved during evolution.