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Introduction: Developmental processes continue in organisms in which sensory systems have reached functional maturity, however, little research has focused on the influence of sensory input on cell and tissue development. Here, we explored the influence of visual system activity on the development of skin melanophores in Xenopus laevis. Methods: Melanophore number was measured in X. laevis larvae after the manipulation of visual input through eye removal (enucleation) and/or incubation on a white or black substrate at the time when the visual system becomes functional (stage 40). To determine the developmental process impacted by visual input, migration, proliferation and differentiation of melanophores was assessed. Finally, the role of melatonin in driving melanophore differentiation was explored. Results: Enucleating, or maintaining stage 40 larvae on a black background, results in a pronounced increase in melanophore number in the perioptic region within 24 h. Time lapse analysis revealed that in enucleated larvae new melanophores appear through gradual increase in pigmentation, suggesting unpigmented cells in the perioptic region differentiate into mature melanophores upon reduced visual input. In support, we observed increased expression of melanization genes tyr, tyrp1, and pmel in the perioptic region of enucleated or black background-reared larvae. Conversely, maintaining larvae in full light suppresses melanophore differentiation. Interestingly, an extra-pineal melatonin signal was found to be sufficient and necessary to promote the transition to differentiated melanophores. Discussion: In this study, we found that at the time when the visual system becomes functional, X. laevis larvae possess a population of undifferentiated melanophores that can respond rapidly to changes in the external light environment by undergoing differentiation. Thus, we propose a novel mechanism of environmental influence where external sensory signals influence cell differentiation in a manner that would favor survival.
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Purpose: Plasmalogens (Plgs) are highly abundant lipids in the retina, and their deficiency leads to severe abnormalities during eye development. The first acylation step in the synthesis of Plgs is catalyzed by the enzyme glyceronephosphate O-acyltransferase (GNPAT), which is also known as dihydroxyacetone phosphate-acyltransferase (EC 2.3.1.42). GNPAT deficiency produces rhizomelic chondrodysplasia punctata type 2, a genetic disorder associated with developmental ocular defects. Despite the relevance of retinal Plgs, our knowledge of the mechanisms that regulate their synthesis, and the role of GNPAT during eye development is limited. Methods: Using the Xenopus laevis model organism, we characterized by in situ hybridization the expression pattern of gnpat and compared it to glycerol 3-phosphate acyltransferase mitochondrial (gpam or gpat1) during eye neurogenesis, lamination, and morphogenesis. The Xenopus Gnpat was biochemically characterized in a heterologous expression system in yeast. Results: During development, gnpat is expressed in proliferative cells of the retina and lens, and post-embryogenesis in proliferative cells of the ciliary marginal zone and lens epithelium. In contrast, gpam expression is mainly restricted to photoreceptors. Xenopus Gnpat expressed in yeast is present in both soluble and membrane fractions, but only the membrane-bound enzyme displays activity. The amino terminal of Gnpat, conserved in humans, shows lipid binding capacity that is enhanced by phosphatidic acid. Conclusions: Enzymes involved in the Plgs and glycerophospholipid biosynthetic pathways are differentially expressed during eye morphogenesis. The gnpat expression pattern and the molecular determinants regulating Gnpat activity advance our knowledge of this enzyme, contributing to our understanding of the retinal pathophysiology associated with GNPAT deficiency.
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Aciltransferases , Plasmalogênios , Proteínas de Xenopus , Animais , Humanos , Aciltransferases/genética , Aciltransferases/metabolismo , Plasmalogênios/metabolismo , Saccharomyces cerevisiae/metabolismo , Xenopus laevis/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Crypsis increases survival by reducing predator detection. Xenopus laevis tadpoles decode light properties from the substrate to induce two responses: a cryptic coloration response where dorsal skin pigmentation is adjusted to the colour of the substrate (background adaptation) and a behavioural crypsis where organisms move to align with a specific colour surface (background preference). Both processes require organisms to detect reflected light from the substrate. We explored the relationship between background adaptation and preference and the light properties able to trigger both responses. We also analysed which retinal photosensor (type II opsin) is involved. Our results showed that these two processes are segregated mechanistically, as there is no correlation between the preference for a specific background with the level of skin pigmentation, and different dorsal retina-localized type II opsins appear to underlie the two crypsis modes. Indeed, inhibition of melanopsin affects background adaptation but not background preference. Instead, we propose pinopsin is the photosensor involved in background preference. pinopsin mRNA is co-expressed with mRNA for the sws1 cone photopigment in dorsally located photoreceptors. Importantly, the developmental onset of pinopsin expression aligns with the emergence of the preference for a white background, but after the background adaptation phenotype appears. Furthermore, white background preference of tadpoles is associated with increased pinopsin expression, a feature that is lost in premetamorphic froglets along with a preference for a white background. Thus, our data show a mechanistic dissociation between background adaptation and background preference, and we suggest melanopsin and pinopsin, respectively, initiate the two responses.
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Opsinas , Opsinas de Bastonetes , Luz , Opsinas/genética , Células Fotorreceptoras , Retina , Opsinas de Bastonetes/genética , Pigmentação da Pele/genéticaRESUMO
Coupling skin colour with the light/dark cycle helps regulate body temperature in ectotherms. In X. laevis, nocturnal release of melatonin from the pineal complex induces pigment aggregation and skin lightening. This nocturnal blanching is initiated by a sensor (type II opsin) that triggers melatonin release when light intensity falls below a minimum threshold, and an effector (melatonin receptor) in the skin which induces pigment aggregation. The sensor/s and effector/s belong to two families of G-protein coupled receptors that originated from a common ancestor, but diverged with subsequent evolution. The aim of this work was to identify candidate sensor/s and effector/s that regulate melatonin-mediated skin colour variation. In X. laevis, we identified a developmental time (stage 43/44) when skin lightening depends on pineal complex photosensitivity alone. At this stage, the pineal complex comprises the frontal organ and pineal gland. A total of 37 type II opsin (14 duplicated) and 6 melatonin receptor (3 duplicated) genes were identified through a full genome analysis of the allotetraploid, X. laevis. These genes were grouped into subfamilies based on their predicted amino acid sequences and the presence of specific amino acids essential for their function. The pineal complex expresses mainly blue light sensitive opsins [pinopsin, parietopsin, opn3, and melanopsins (opn4 and opn4b)] and UV-light sensitive opsins (opn5 and parapinopsin), while visual opsins and va-ancient opsin are absent, as determined by RT-PCR and in situ hybridization. The photoisomerase retinal G-protein coupled receptor, and an uncharacterized opn6b opsin, are also expressed. The spectral sensitivity that triggers melatonin secretion, and therefore melanophore aggregation, falls in the visible spectrum (470-650 ηm) and peaks in the blue/green range, pointing to the involvement of opsins with sensitivities therein. The effector-melatonin receptors expressed in skin melanophores are mtnr1a and mtnr1c. Our data point to candidate proteins required in the neuroendocrine circuit that underlies the circadian regulation of skin pigmentation, and suggest that multiple initiators and effectors likely participate.
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Meio Ambiente , Luz , Melanóforos/metabolismo , Melanóforos/efeitos da radiação , Opsinas/metabolismo , Receptores de Melatonina/metabolismo , Pigmentação da Pele/efeitos da radiação , Sequência de Aminoácidos , Animais , Opsinas/química , Xenopus laevisRESUMO
Lower vertebrates use rapid light-regulated changes in skin colour for camouflage (background adaptation) or during circadian variation in irradiance levels. Two neuroendocrine systems, the eye/alpha-melanocyte-stimulating hormone (α-MSH) and the pineal complex/melatonin circuits, regulate the process through their respective dispersion and aggregation of pigment granules (melanosomes) in skin melanophores. During development, Xenopus laevis tadpoles raised on a black background or in the dark perceive less light sensed by the eye and darken in response to increased α-MSH secretion. As embryogenesis proceeds, the pineal complex/melatonin circuit becomes the dominant regulator in the dark and induces lightening of the skin of larvae. The eye/α-MSH circuit continues to mediate darkening of embryos on a black background, but we propose the circuit is shut down in complete darkness in part by melatonin acting on receptors expressed by pituitary cells to inhibit the expression of pomc, the precursor of α-MSH.
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Luz , Sistemas Neurossecretores/metabolismo , Sistemas Neurossecretores/efeitos da radiação , Pigmentação da Pele/efeitos da radiação , Animais , Escuridão , Embrião não Mamífero/metabolismo , Embrião não Mamífero/efeitos da radiação , Desenvolvimento Embrionário/efeitos da radiação , Cinética , Larva/efeitos da radiação , Melanóforos/metabolismo , Melanóforos/efeitos da radiação , Melatonina/metabolismo , Hipófise/metabolismo , Pró-Opiomelanocortina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Xenopus laevis/embriologia , alfa-MSH/metabolismoRESUMO
BACKGROUND: Antioxidants such as the green tea polyphenol epigallocatechin gallate (EGCG) are neuroprotective under many conditions in mature nervous systems; however, their impact has rarely been explored in developing nervous systems, in which a critical step is the formation of connections between neurons. Axons emerge from newly formed neurons and are led by a dynamic structure found at their tip called a growth cone. Here we explore the impact of EGCG on the development of retinal ganglion cell (RGC) axons, which connect the eye to the brain. RESULTS: EGCG acts directly on RGC axons to increase the number of growth cone filopodia, fingerlike projections that respond to extrinsic signals, in vitro and in vivo. Furthermore, EGCG exposure leads to a dramatic defect in the guided growth of RGC axons where the axons fail to make a key turn in the mid-diencephalon required to reach their target. Intriguingly, at guidance points where RGCs do not show a change in direction, EGCG has no influence on RGC axon behavior. CONCLUSIONS: We propose that EGCG stabilizes filopodia and prevents normal filopodial dynamics required for axons to change their direction of outgrowth at guidance decision points. Developmental Dynamics 245:667-677, 2016. © 2016 Wiley Periodicals, Inc.
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Axônios/efeitos dos fármacos , Axônios/metabolismo , Catequina/análogos & derivados , Cones de Crescimento/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Orientação de Axônios/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Catequina/farmacologia , Gonadotropina Coriônica/farmacologia , Diencéfalo/citologia , Diencéfalo/efeitos dos fármacos , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Feminino , Humanos , Hibridização In Situ , Neurogênese/efeitos dos fármacos , Retina/citologia , Retina/efeitos dos fármacos , XenopusRESUMO
Neurons acquire a unique cell-type dependent morphology during development that is critical for their function in a neural circuit. The process involves a neuron sending out an axon that grows in a directed fashion to its target, and the elaboration of multiple, branched dendrites. The ultimate morphology of the neuron is sculpted by factors in the environment that act directly or indirectly to influence the behavior of the growing axon and dendrites. The output neuron of the retina, the retinal ganglion cell (RGC), has served as a useful model for the identification of molecular signals that control neuronal morphogenesis, because the entire development of the neuron, from the initiation of neurites to the establishment of synapses, is accessible for experimental manipulation and visualization. In this review we discuss data which argue that the visual system uses a limited number of signals to control RGC morphogenesis, with single molecules being reused multiple times to control distinct events in axon and dendrite outgrowth.
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Morfogênese/fisiologia , Retina/citologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologia , Animais , Axônios/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Dendritos/fisiologia , Morfogênese/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Vias Visuais/anatomia & histologia , Vias Visuais/embriologia , Vias Visuais/crescimento & desenvolvimentoRESUMO
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.
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Axônios/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Semaforina-3A/metabolismo , Transdução de Sinais/fisiologia , Vias Visuais/anatomia & histologia , Animais , Diencéfalo/citologia , Diencéfalo/metabolismo , Embrião não Mamífero , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/farmacologia , Pirróis/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Células Ganglionares da Retina/citologia , Semaforina-3A/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Vias Visuais/embriologia , Vias Visuais/metabolismo , XenopusRESUMO
The role of extrinsic cues in guiding developing axons is well established; however, the means by which the activity of these extrinsic cues is regulated is poorly understood. A disintegrin and metalloproteinase (ADAM) enzymes are Zn-dependent proteinases that can cleave guidance cues or their receptors in vitro. Here, we identify the first example of a metalloproteinase that functions in vertebrate axon guidance in vivo. Specifically, ADAM10 is required for formation of the optic projection by Xenopus retinal ganglion cell (RGC) axons. Xadam10 mRNA is expressed in the dorsal neuroepithelium through which RGC axons extend. Pharmacological or molecular inhibition of ADAM10 within the brain each resulted in a failure of RGC axons to recognize their target. In contrast, molecular inhibition of ADAM10 within the RGC axons themselves had no effect. These data argue strongly that in the dorsal brain ADAM10 acts cell non-autonomously to regulate the guidance of RGC axons.
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Proteínas ADAM/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Axônios/enzimologia , Sistemas de Liberação de Medicamentos , Proteínas de Membrana/fisiologia , Retina/enzimologia , Células Ganglionares da Retina/enzimologia , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Animais , Axônios/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Inibidores de Proteases/administração & dosagem , Retina/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Xenopus laevisRESUMO
A learning event can be dissociated into 3 components: acquisition, storage, and recall. When the laboratory wild-type strain of Caenorhabditis elegans (N2 strain) is exposed to benzaldehyde in the absence of food, the worms display a reduction of their attractive response to this volatile odorant. This results from the association between benzaldehyde and a nutrient-deficient environment. Another wild-type isolate, the CB4856 strain, fails to display this decreased response to benzaldehyde after exposure to benzaldehyde in the absence of food. However, like the N2 strain, when tested to isoamyl alcohol after benzaldehyde conditioning, the CB4856 strain displays a decreased isoamyl alcohol response. Therefore, the CB4856 strain does not have an acquisition deficit, but it suffers from a recall deficit specific to benzaldehyde.
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Caenorhabditis elegans/fisiologia , Aprendizagem/fisiologia , Rememoração Mental/fisiologia , Adaptação Fisiológica/fisiologia , Animais , Comportamento Animal , Benzaldeídos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Fatores Quimiotáticos/farmacologia , Quimiotaxia/fisiologia , Diacetil/farmacologia , Relação Dose-Resposta a Droga , Aprendizagem/efeitos dos fármacos , Deficiências da Aprendizagem/genética , Rememoração Mental/efeitos dos fármacos , Mutação , Odorantes , Pentanóis/farmacologia , Especificidade da Espécie , InaniçãoRESUMO
We demonstrate that Caenorhabditis elegans is able to form an association between the presence of the odorant benzaldehyde and the food content of its environment. When exposed to 100% benzaldehyde for 1 h in the absence of food the naive attractive response is reduced, and we have found that this olfactory adaptation is attenuated by the presence of food. Contrary to nonassociative (single stimulus) learning theory, this response is not a function of the total time of exposure to benzaldehyde but rather an associative function of the ability of benzaldehyde to predict a nutrient-deficient environment. Genetic and pharmacological evidence revealed that the effects of food in this learning paradigm are mediated by serotonergic signaling.