Axons are guided to their targets by molecular cues expressed in their environment. How is the presence of these cues regulated? Although some evidence indicates that morphogens establish guidance cue expression as part of their role in patterning tissues, an important question is whether morphogens are then required to maintain guidance signals. We found that fibroblast growth factor (FGF) signaling sustains the expression of two guidance cues, semaphorin3A (xsema3A) and slit1 (xslit1), throughout the period of Xenopus optic tract development. With FGF receptor inhibition, xsema3A and xslit1 levels were rapidly diminished, and retinal ganglion cell axons arrested in the mid-diencephalon, before reaching their target. Importantly, direct downregulation of XSema3A and XSlit1 mostly phenocopied this axon guidance defect. Thus, FGFs promote continued presence of specific guidance cues critical for normal optic tract development, suggesting a second later role for morphogens, independent of tissue patterning, in maintaining select cues by acting to regulate their transcription.

How skin colour adjusts to circadian light/dark cycles is poorly understood. Melanopsin (Opn4) is expressed in melanophores, where in vitro studies suggest it regulates skin pigmentation through a 'primary colour response' in which light photosensitivity is translated directly into pigment movement. However, the entrainment of the circadian rhythm is regulated by a population of melanopsin-expressing retinal ganglion cells (mRGCs) in the eye. Therefore, in vivo, melanopsin may trigger a 'secondary colour response' initiated in the eye and controlled by the neuro-endocrine system. We analysed the expression of opn4m and opn4x and melanin aggregation induced by light (background adaptation) in Xenopus laevis embryos. While opn4m and opn4x are expressed at early developmental times, light-induced pigment aggregation requires the eye to become functional. Pharmacological inhibition of melanopsin suggests a model whereby mRGC activation lightens skin pigmentation via a secondary response involving negative regulation of alpha-melanocyte-stimulating hormone (α-MSH) secretion by the pituitary.

We show that mitogenic cells expressing T-type Ca 2ϩ channels (T-channels) are more sensitive to the antiproliferative effects of the drugs pimozide and mibefradil than cells without significant T-channel expression. The growth of Y79 and WERI-Rb1 retinoblastoma cells, as well as MCF7 breast cancer epithelial cells, all of which express T-channel current and mRNA for T-channel subunits, is inhibited by pimozide and mibefradil with IC 50 values between 0.6 and 1.5 M. Proliferation of glioma C6 cells, which show little T-channel expression, is less sensitive to these drugs (IC 50 ϭ 8 and 5 M for pimozide and mibefradil, respectively). Neither drug seems to alter cell cycle or the expression of cyclins. Although this strong correlation between T-channel expression and growth inhibition exists, the following results suggest that the drugs inhibit cell growth via different cytotoxic pathways: 1) pimozide and mibefradil have additive effects on T-channel current inhibition, whereas the antiproliferative activity of the drugs together is synergistic; 2) an increase in the number of apoptotic Y79 and MCF7 cells and a decrease in the mRNA for the antiapoptotic gene Bcl-2 is detected only in pimozide-treated cells, whereas in mibefradiltreated cells, the toxicity is primarily necrotic; and 3) growth inhibition by mibefradil is reduced in Y79 cells transfected with T-channel antisense and in differentiated Y79 cells (which have decreased T-channel expression), but growth inhibition by pimozide is affected to a lesser extent. These results suggest that pimozide and mibefradil inhibit cell proliferation via different cytotoxic pathways and that in the case of pimozide, it is unlikely that this effect is mediated solely by T-channel inhibition.

BackgroundLight information is sorted by neuronal circuits to generate image-forming (IF) (interpretation and tracking of visual objects and patterns) and non-image-forming (NIF) tasks. Among the NIF tasks, photic entrainment of circadian rhythms, the pupillary light reflex, and sleep are all associated with physiological responses, mediated mainly by a small group of melanopsin-expressing retinal ganglion cells (mRGCs). Using Xenopus laevis as a model system, and analyzing the c-fos expression induced by light as a surrogate marker of neural activity, we aimed to establish the developmental time at which the cells participating in both systems come on-line in the retina.ResultsWe found that the peripheral retina contains 80% of the two melanopsin-expressing cell types we identified in Xenopus: melanopsin-expressing horizontal cells (mHCs; opn4m+/opn4x+/Prox1+) and mRGCs (2.7% of the total RGCs; opn4m+/opn4x+/Pax6+/Isl1), in a ratio of 6:1. Only mRGCs induced c-fos expression in response to light. Dopaminergic (tyrosine hydroxylase-positive; TH+) amacrine cells (ACs) may be part of the melanopsin-mediated circuit, as shown by preferential c-fos induction by blue light. In the central retina, two cell types in the inner nuclear layer (INL) showed light-mediated induction of c-fos expression [(On-bipolar cells (Otx2+/Isl1+), and a sub-population of ACs (Pax6−/Isl1−)], as well as two RGC sub-populations (Isl1+/Pax6+ and Isl1+/Pax6−). Melanopsin and opsin expression turned on a day before the point at which c-fos expression could first be activated by light (Stage 37/38), in cells of both the classic vision circuit, and those that participate in the retinal component of the NIF circuit. Key to the classic vision circuit is that the component cells engage from the beginning as functional ‘unit circuits’ of two to three cells in the INL for every RGC, with subsequent growth of the vision circuit occurring by the wiring in of more units.ConclusionsWe identified melanopsin-expressing cells and specific cell types in the INL and the RGC layer which induce c-fos expression in response to light, and we determined the developmental time when they become active. We suggest an initial formulation of retinal circuits corresponding to the classic vision pathway and melanopsin-mediated circuits to which they may contribute.

Summary Melanopsin photopigments, Opn4x and Opn4m, were evolutionary selected to “see the light” in systems that regulate skin colour change. In this review, we analyse the roles of melanopsins, and how critical evolutionary developments, including the requirement for thermoregulation and ultraviolet protection, the emergence of a background adaptation mechanism in land‐dwelling amphibian ancestors and the loss of a photosensitive pineal gland in mammals, may have helped sculpt the mechanisms that regulate light‐controlled skin pigmentation. These mechanisms include melanopsin in skin pigment cells directly inducing skin darkening for thermoregulation/ultraviolet protection; melanopsin‐expressing eye cells controlling neuroendocrine circuits to mediate background adaptation in amphibians in response to surface‐reflected light; and pineal gland secretion of melatonin phased to environmental illuminance to regulate circadian and seasonal variation in skin colour, a process initiated by melanopsin‐expressing eye cells in mammals, and by as yet unknown non‐visual opsins in the pineal gland of non‐mammals.