The development and pattern of innervation of the avian cornea

The involvement of nerves in the development of the avian cornea is poorly understood, primarily because the demonstration of corneal nerves has proved to be elusive. In the present study, the development of corneal innervation is demonstrated by the application of a modified Bodian staining technique (J. Lewis, 1978, Zoon, 6, 175–179). On the 6th day of embryonic development, numerous large fascicles of axons are observed arriving at the ventrotemporal aspect of the cornea, within the periocular mesenchyme. These fascicles subdivide into two distinct groups which migrate both ventrally and, more extensively, dorsally around the cornea. Progressive migration of nerve fascicles around the cornea occurs through the 7th and 8th days of development, and by the 10th day the cornea is ensheathed within a ring of nerves. Concomitant with ring formation, nerves are observed leaving the main nerve fascicles and migrating toward the cornea. Numerous nerve processes, which enter through the mid-stroma, are observed migrating toward the center of the 12th-day cornea. Innervation of the epithelium is detected on the 12th day, beginning at the periphery and increasing dramatically with development. Innervation of the epithelium is almost complete on the 16th day and penetration of nerves into the central stroma occurs on the 18th day of development. On the 16th day, the basal epithelial cells begin to demonstrate silver-staining properties. The levels of this staining increase with development, and in the hatchling the squamous cells demonstrate a characteristic silver-staining pattern. Innervation of the corneal endothelium is not observed. These results indicate that the avian cornea and its epithelium become innervated over the same developmental period in which the major transition from corneal opacity to transparency is achieved.     

Cholinergic innervation of the cornea and iris of the guinea pig

Cholinergic innervation of the cornea and iris of the newborn and adult guinea pig was studied by the technique of Karnovsky and Roots (1964). The given structures are both richly innervated. The cholinesterase reaction of the cornea is more strongly positive in adult animals, whereas the intensity of the reaction of the iris in newborn and adult guinea pigs is almost identical.     

Lens-derived Semaphorin3A regulates sensory innervation of the cornea

The cornea, one of the most highly innervated tissues of the body, is innervated by trigeminal sensory afferents. During development, axons are initially repelled at the corneal margin, resulting in the formation of a circumferential nerve ring. The nature and source of guidance molecules that regulate this process remain a mystery. Here, we show that the lens, which immediately underlies the cornea, repels trigeminal axons in vivo and in vitro. Lens ablation results in premature, disorganized corneal innervation and disruption of the nerve ring and ventral plexus. We show that Semaphorin3A (Sema3A) is expressed in the lens epithelium and its receptor Neuropilin-1 (Npn1) is expressed in the trigeminal ganglion during cornea development. Inhibition of Sema3A signaling abrogates axon repulsion by the lens and cornea in vitro and phenocopies lens removal in vivo. These results demonstrate that lens-derived Sema3A mediates initial repulsion of trigeminal sensory axons from the cornea and is necessary for the proper formation of the nerve ring and positioning of the ventral plexus in the choroid fissure.     

Nerve repulsion by the lens and cornea during cornea innervation is dependent on Robo-Slit signaling and diminishes with neuron age

The cornea, the most densely innervated tissue on the surface of the body, becomes innervated in a series of highly coordinated developmental events. During cornea development, chick trigeminal nerve growth cones reach the cornea margin at embryonic day (E)5, where they are initially repelled for days from E5 to E8, instead encircling the corneal periphery in a nerve ring prior to entering on E9. The molecular events coordinating growth cone guidance during cornea development are poorly understood. Here we evaluated a potential role for the Robo-Slit nerve guidance family. We found that Slits 1, 2 and 3 expression in the cornea and lens persisted during all stages of cornea innervation examined. Robo1 expression was developmentally regulated in trigeminal cell bodies, expressed robustly during nerve ring formation (E5-8), then later declining concurrent with projection of growth cones into the cornea. In this study we provide in vivo and in vitro evidence that Robo-Slit signaling guides trigeminal nerves during cornea innervation. Transient, localized inhibition of Robo-Slit signaling, by means of beads loaded with inhibitory Robo-Fc protein implanted into the developing eyefield in vivo, led to disorganized nerve ring formation and premature cornea innervation. Additionally, when trigeminal explants (source of neurons) were oriented adjacent to lens vesicles or corneas (source of repellant molecules) in organotypic tissue culture both lens and cornea tissues strongly repelled E7 trigeminal neurites, except in the presence of inhibitory Robo-Fc protein. In contrast, E10 trigeminal neurites were not as strongly repelled by cornea, and presence of Robo-Slit inhibitory protein had no effect. In full, these findings suggest that nerve repulsion from the lens and cornea during nerve ring formation is mediated by Robo-Slit signaling. Later, a shift in nerve guidance behavior occurs, in part due to molecular changes in trigeminal neurons, including Robo1 downregulation, thus allowing nerves to find the Slit-expressing cornea permissive for growth cones.     

Observations on the morphology of the developing primate cornea: epithelium, its innervation and anterior stroma.

A survey is made of some ultrastructural features of the developing cornea of Macaca mulatta. The observations are confined to the anterior central area, starting with the lens vesicle stage and progressing through midgestation, when the morphologic characteristics of the cornea are fully established. Subepithelial filaments and some partially aggregated collagen fibrils are present in the earliest embryo and are of a size and appearance similar to those in the future vitreous cavity. Epithelial secretory activity points to, but does not prove direct contribution to the deposition of the acellular matrix components beneath it. No trace of a structured, orthogonal collagenous stroma can be visualized. The primitive endothelium forms prior to the fibroblast invasion of the distended filamentous matrix. Bowman's layer has undoubted epithelial contributions. Its aggregated collagen fibrils have approximately the same diameter as those of the anterior stroma. Intraepithelial appearance of single nerve fibers and fascicles takes place during the first trimester of gestation, as soon as the two continuous epithelial layers are formed. Terminal areas approach closely to the basal cell's nucleus, without touching it. The plasmalemma of the invaginating nerve fiber is surrounded by that of the epithelial cell in a mesaxon-like manner, with occasional gap junctions uniting adjoining neural and epithelial cell membranes. The fetal neurites contain microtubules, some clear vesicles and dense vacuoles resembling those of mature monamine and non-monamine neurons. Mitochondria are small and compact, their presence indicating a high rate of metabolic activity in the immature terminal area.     

Lens formation from the cornea following implantation into hindlimbs of larval Xenopus laevis: the influence of limb innervation and extent of differentiation

Corneal fragments of larval Xenopus laevis at stage 48 (according to Nieuwkoop and Faber, '56), were implanted into sham denervated unamputated hindlimbs, denervated unamputated hindlimbs, amputated and sham denervated hindlimbs, and amputated and denervated hindlimbs of larvae at stages 52 and 57. The results show that unamputated limbs at stage 52, either innervated or denervated, manifest a weak capacity to promote the first lens-forming transformations of the outer cornea. This capacity is absent in both limb types at stage 57. After amputation, limbs of both early and late stages form a regenerative blastema and support lens formation from the outer cornea. Denervation of early stage limbs has no appreciable effect on blastema formation and lens-forming transformation of corneal implants. However, denervation of late stage limbs inhibits both processes. These results indicate that the limb tissues of the early stage limbs contain non-neural inductive factors at a low level and that after limb amputation and blastema formation the level of these factors becomes high enough to promote lens formation from implanted cornea, even after denervation. In contrast, the limb tissues of late stage limbs do not contain a suitable level of non-neural inductive factors.     

Lens formation from cornea implanted into amputated hindlimbs of Xenopus laevis larvae requires innervation or proliferating cell populations in the stump

The capacity of amputated early and late limbs of larval Xenopus laevis to promote lens-forming transformations of corneal implants in the absence of a limb regeneration blastema has been tested by implanting outer cornea fragments from donor larvae at stage 48 (according to Nieuwkoop and Faber 1956), into limb stumps of larvae at stage 52 and 57. Blastema formation has been prevented either by covering the amputation surface with the skin or by reconnecting the amputated part to the limb stump. Results show that stage 52 non-regenerating limbs could promote lens formation from corneal implants not only when innervated but also when denervated. A similar result was observed in stage 57 limbs where blastema formation was prevented by reconnecting the amputated part to the stump. In this case, relevant tissue dedifferentiation was observed in the boundary region between the stump and the autografted part of the limb. However, stage 57 limbs, where blastema formation was prevented by covering the amputation surface with skin, could promote lens formation from the outer cornea only when innervated. In this case, no relevant dedifferentiation of the stump tissues was observed. These results indicate that blastema formation is not a prerequisite for lens-forming transformations of corneal fragments implanted into amputated hindlimbs of larval X. laevis and that lens formation can be promoted by factors delivered by the nerve fibres or produced by populations of undifferentiated or dedifferentiated limb cells.