Bret M. Lehman

The embryonic chick has the ability to regenerate its retina after it has been completely removed. Here, we provide a detailed characterization of retina regeneration in the embryonic chick at the cellular level. Retina regeneration can occur in two distinct manners. The first is via transdifferentiation, which is induced by members of the Fibroblast growth factor (Fgf) family. The second type of retinal regeneration occurs from the anterior margin of the eye, near the ciliary body (CB) and ciliary marginal zone (CMZ). We show that regeneration from the CB/CMZ is the result of proliferating stem/progenitor cells. This type of regeneration is also stimulated by Fgf2, but we show that it can be activated by Sonic hedgehog (Shh) overexpression when no ectopic Fgf2 is present. Shh-stimulated activation of CB/CMZ regeneration is inhibited by the Fgf receptor (Fgfr) antagonist, PD173074. This indicates that Shh-induced regeneration acts through the Fgf signaling pathway. In addition, we show that the hedgehog (Hh) pathway plays a role in maintenance of the retina pigmented epithelium (RPE), as ectopic Shh expression inhibits transdifferentiation and Hh inhibition increases the transdifferentiation domain. Ectopic Shh expression in the regenerating retina also results in a decrease in the number of ganglion cells present and an increase in apoptosis mostly in the presumptive ganglion cell layer (GCL). However, Hh inhibition increases the number of ganglion cells but does not have an effect on cell death. Taken together, our results suggest that the hedgehog pathway is an important modulator of retina regeneration.

BACKGROUND: Many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. Accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. Cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase (ChAT). RESULTS: We found that as ChAT-immunoreactive cells differentiate they expressed the homeodomain transcription factors Pax6 and Islet1, and the cell-cycle inhibitor p27kip1. As differentiation proceeds, type-II cholinergic cells, displaced to the ganglion cell layer, transiently expressed high levels of cellular retinoic acid binding protein (CRABP) and neurofilament, while type-I cells in the inner nuclear layer did not. Although there is a 1:1 ratio of type-I to type-II cells in vivo, in dissociated cell cultures the type-I cells (ChAT-positive and CRABP-negative) out-numbered the type-II cells (ChAT and CRABP-positive cells) by 2:1. The relative abundance of type-I to type-II cells was not influenced by Sonic Hedgehog (Shh), but was affected by compounds that act at muscarinic acetylcholine receptors. In addition, the abundance and mosaic patterning of type-II cholinergic amacrine cells is disrupted by interfering with muscarinic signaling. CONCLUSION: We conclude that: (1) during development type-I and type-II cholinergic amacrine cells are not homotypic, (2) the phenotypic differences between these subtypes of cells is controlled by the local microenvironment, and (3) appropriate levels of muscarinic signaling between the cholinergic amacrine cells are required for proper mosaic patterning.

PURPOSE: To evaluate the validity and repeatability of crystalline lens thickness measurements obtained by anterior segment optical coherence tomography (OCT). METHODS: Forty-seven normal children (mean age, 11.06 +/- 2.30 yr) had their crystalline lens thickness measured with the Visante anterior segment OCT (Carl Zeiss Meditec, Dublin, CA) and with conventional corneal touch A-scan ultransonography (ultrasound) (Humphrey 820). The subjects' right corneas were anesthetized, and their right eyes were cyclopleged. Five ultrasound measurements were recorded per eye, and three Visante OCT measurements were recorded per eye. Thirty-eight subjects had measurements at a second visit where three additional Visante OCT measurements were recorded. RESULTS: The mean of the differences between the Visante OCT and ultrasound was -0.045 mm (p = 0.017) with 95% limits of agreement from -0.29 to 0.20 mm, indicating that the measurement of crystalline lens thickness was slightly thinner with the Visante OCT. When validity was assessed using only Visante OCT images that contained the corneal reflex, the mean of the differences was 0.019 mm (p = 0.11) with 95% limits of agreement from -0.091 to 0.13 mm. For the repeatability of the Visante OCT, the mean of the differences between visit one and visit two was -0.008 mm (p = 0.25) with 95% limits of agreement from -0.088 to 0.072 mm. Repeatability improved when reassessed using only images that contain the corneal reflex; the mean of the differences was -0.0001 mm (p = 0.97) with 95% limits of agreement from -0.030 to 0.030 mm. CONCLUSION: The Visante OCT is a non-contact instrument that is simple to use, and it provides valid crystalline lens thickness measurements with excellent repeatability. Validity and repeatability are optimized when the Visante OCT images contain the corneal reflex and a consistent corneal index refraction is applied to the entire image.