Embryonic origin of avian corneal sensory nerves.

Sensory nerves play a vital role in maintaining corneal transparency. They originate in the trigeminal ganglion, which is derived from two embryonic cell populations (cranial neural crest and ectodermal placode). Nonetheless, it is unclear whether corneal nerves arise from neural crest, from placode, or from both. Quail-chick chimeras and species-specific antibodies allowed tracing quail-derived neural crest or placode cells during trigeminal ganglion and corneal development, and after ablation of either neural crest or placode. Neural crest chimeras showed quail nuclei in the proximal part of the trigeminal ganglion, and quail nerves in the pericorneal nerve ring and in the cornea. In sharp contrast, placode chimeras showed quail nuclei in the distal part of the trigeminal ganglion, but no quail nerves in the cornea or in the pericorneal nerve ring. Quail placode-derived nerves were present, however, in the eyelids. Neural crest ablation between stages 8 and 9 resulted in diminished trigeminal ganglia and absence of corneal innervation. Ablation of placode after stage 11 resulted in loss of the ophthalmic branch of the trigeminal ganglion and reduced corneal innervation. Noninnervated corneas still became transparent. These results indicate for the first time that although both neural crest and placode contribute to the trigeminal ganglion, corneal innervation is entirely neural crest-derived. Nonetheless, proper corneal innervation requires presence of both cell types in the embryonic trigeminal ganglion. Also, complete lack of innervation has no discernible effect on development of corneal transparency or cell densities.

Specificity of corneal nerve positions during embryogenesis.

PURPOSE: Embryonic corneal innervation first involves pericorneal nerve ring formation, with nerves in specific positions, followed by innervation of the corneal stroma from the ring. Here we determine whether nerve bundles enter the cornea at specific locations along the ring and whether bundles enter the cornea at specific depths. METHODS: Whole mount embryonic quail corneas immunostained for nerves were scanned using confocal laser microscopy. Images were superimposed digitally in pseudo-colored pairs to detect similar positions of innervation, and then rotated stepwise to determine if degree of synchrony was decreased. Degrees of innervation of each corneal quadrant were quantified. Depths of stromal bundles innervating the cornea were determined by depth of focus analysis. RESULTS: Superimposition of images indicated many nerve entry points in similar locations, suggesting specificity. However, stepwise rotations of one image above the other revealed that degree of positional synchrony remained constant, suggesting that nerves do not occur in specific locations, but rather simply at approximately equal distances around the cornea. Corneas from both left and right sides are innervated by similar numbers of nerve bundles (Left, 44+/-0.4; Right, 44+/-1.0), with the same number/quadrant (Left, 11+/-0.2; Right, 11+/-0.2). Nerves entering the stroma closest to Descemet's layer innervate either the entire cornea along that radius or only the central-peripheral and central cornea; those entering nearer Bowman's layer innervate only peripheral cornea. CONCLUSIONS: Avian corneal nerve bundles enter along radii spaced at equal intervals along the pericorneal nerve ring, suggesting an innervation mechanism based on equal spacing between nerves. Nerve bundles from the nerve ring enter the stroma at depths correlated with their subsequent targets.

Simulated microgravity and hypergravity attenuate heart tissue development in explant culture.

Exposure to altered gravity may disturb the cytoskeleton-cell surface-extracellular matrix (ECM) interface of embryonic cells. Development of organs such as the heart depends on dynamic interactions across cell surfaces. Fibronectin (FN), for example, a glycoprotein that links the ECM to the cytoskeleton through integrin surface receptors, is required for normal heart development. Thus, altered gravity may perturb organogenesis. We cultured precardiac explants from chick embryos in a rotating bioreactor vessel to simulate microgravity (microG), or in a tissue culture centrifuge, for 18 h during heart development. Bioreactor microG did not alter external morphology of explants, but did significantly reduce the proportion that developed contractions. Immunostaining for FN of explant sections showed that it also significantly reduced the linear extent of staining present in basement membrane regions. Analysis of ultrastructure revealed a significant reduction in the number of desmosomes per unit area and other differences. Hypergravity dramatically abolished development of contractions and altered morphogenesis. The results indicate a probable sensitivity of cardiomyogenic development involving FN to altered gravity.

Nuclear morphologies of bovine corneal cells as visualized by confocal microscopy.

Confocal laser scanning microscopy was used to characterize nuclear morphology of the three cell layers of bovine cornea in vivo. Corneas fixed with formalin were stained with propidium iodide, whereas living cells in nonfixed corneas were stained with PicoGreen. Nuclei in the three corneal cell layers consistently assume strikingly different shapes. Round nuclei were observed throughout the layers of the epithelium of both fixed and living cells. Stromal fibroblasts (keratocytes) showed approximately equal numbers of elliptical and bean-shaped nuclei arrayed in a variety of orientations. Keratocytes near Bowman's layer had almost round nuclei whereas those near Descemet's membrane had more elongated elliptical nuclei. Lobulate nuclei arranged in a regular pattern were observed throughout the endothelium. Some of the lobulate nuclei were large and stained less intensely with the fluorescent dyes. In addition, keratocytes in vitro displayed the same two distinct nuclear morphologies as in vivo. These observations indicate that each cell layer of the cornea contains nuclei with characteristic morphologies and that, in the case of keratocytes, the cells maintain their characteristic nuclear morphologies in vitro.