Cell-type expression and activation by light of neuropsins in the developing and mature Xenopus retina.

Photosensitive opsins detect light and perform image- or nonimage-forming tasks. Opsins such as the "classical" visual opsins and melanopsin are well studied. However, the retinal expression and functions of a novel family of neuropsins are poorly understood. We explored the developmental time-course and cell-type specificity of neuropsin (opn5, 6a, 6b, and 8) expression in Xenopus laevis by in situ hybridization and immunohistochemistry. We compared the Xenopus results with publicly available single cell RNA sequencing (scRNA-seq) data from zebrafish, chicken, and mouse. Additionally, we analyzed light-activation of neuropsin-expressing cells through induction of c-fos mRNA. opn5 and opn8 expression begins at stage 37/38 when the retinal circuits begin to be activated. Once retinal circuits connect to the brain, opn5 mRNA is distributed across multiple retinal cell types, including bipolar (~70%-75%), amacrine (~10%), and retinal ganglion (~20%) cells, with opn8 present in amacrine (~70%) and retinal ganglion (~30%) cells. opn6a and opn6b mRNAs emerge in newborn-photoreceptors (stage 35), and are colocalized in rods and cones by stage 37/38. Interestingly, in the mature larval retina (stage 43/44), opn6a and opn6b mRNAs become preferentially localized to rods and cones, respectively, while newborn photoreceptors bordering the proliferative ciliary marginal zone express both genes. In zebrafish, opn6a and opn6b are also expressed in photoreceptors, while Müller glia and amacrine cells express opn8c. Most neuropsin-expressing retinal ganglion cells display c-fos expression in response to light, as do over half of the neuropsin-expressing interneurons. This study gave a better understanding of retinal neuropsin-expressing cells, their developmental onset, and light activation.

Differential Eye Expression of Xenopus Acyltransferase Gnpat and Its Biochemical Characterization Shed Light on Lipid-Associated Ocular Pathologies.

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.

TRPM8 thermosensation in poikilotherms mediates both skin colour and locomotor performance responses to cold temperature.

Thermoregulation is a homeostatic process to maintain an organism's internal temperature within a physiological range compatible with life. In poikilotherms, body temperature fluctuates with that of the environment, with both physiological and behavioral responses employed to modify body temperature. Changing skin colour/reflectance and locomotor activity are both well-recognized temperature regulatory mechanisms, but little is known of the participating thermosensor/s. We find that Xenopus laevis tadpoles put in the cold exhibit a temperature-dependent, systemic, and rapid melanosome aggregation in melanophores, which lightens the skin. Cooling also induces a reduction in the locomotor performance. To identify the cold-sensor, we focus on transient receptor potential (trp) channel genes from a Trpm family. mRNAs for several Trpms are present in Xenopus tails, and Trpm8 protein is present in skin melanophores. Temperature-induced melanosome aggregation is mimicked by the Trpm8 agonist menthol (WS12) and blocked by a Trpm8 antagonist. The degree of skin lightening induced by cooling is correlated with locomotor performance, and both responses are rapidly regulated in a dose-dependent and correlated manner by the WS12 Trpm8 agonist. We propose that TRPM8 serves as a cool thermosensor in poikilotherms that helps coordinate skin lightening and behavioural locomotor performance as adaptive thermoregulatory responses to cold.

Distinct type II opsins in the eye decode light properties for background adaptation and behavioural background preference.

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.

Type II Opsins in the Eye, the Pineal Complex and the Skin of Xenopus laevis: Using Changes in Skin Pigmentation as a Readout of Visual and Circadian Activity.

The eye, the pineal complex and the skin are important photosensitive organs. The African clawed frog, Xenopus laevis, senses light from the environment and adjusts skin color accordingly. For example, light reflected from the surface induces camouflage through background adaptation while light from above produces circadian variation in skin pigmentation. During embryogenesis, background adaptation, and circadian skin variation are segregated responses regulated by the secretion of α-melanocyte-stimulating hormone (α-MSH) and melatonin through the photosensitivity of the eye and pineal complex, respectively. Changes in the color of skin pigmentation have been used as a readout of biochemical and physiological processes since the initial purification of pineal melatonin from pigs, and more recently have been employed to better understand the neuroendocrine circuit that regulates background adaptation. The identification of 37 type II opsin genes in the genome of the allotetraploid X. laevis, combined with analysis of their expression in the eye, pineal complex and skin, is contributing to the elucidation of the role of opsins in the different photosensitive organs, but also brings new questions and challenges. In this review, we analyze new findings regarding the anatomical localization and functions of type II opsins in sensing light. The contribution of X. laevis in revealing the neuroendocrine circuits that regulate background adaptation and circadian light variation through changes in skin pigmentation is discussed. Finally, the presence of opsins in X. laevis skin melanophores is presented and compared with the secretory melanocytes of birds and mammals.

Melanin-concentrating hormone like and somatolactin. A teleost-specific hypothalamic-hypophyseal axis system linking physiological and morphological pigmentation.

Plastic adaptation to match the skin colour to the surrounding is key to survival. Two biological responses in skin colour are associated with background adaptation. A fast "physiological response" that aggregates/disperses the pigment organelles of skin chromatophores, and a slow "morphological response" that alters the type and/or density of pigment cells in the skin. Both responses are linked by unknown mechanisms. In this review, we discuss the role in skin colour regulation of two molecules that form part of a hypothalamic-hypophyseal pathway unique to teleosts, melanin-concentrating hormone "like" (MCHL) (previously known as MCH), and somatolactin. MCHL neurons project to the neurohypophysis and to the pars intermedia pituitary, where they interact with somatolactin-expressing cells. With a white background MCHL is released into the circulation to induce rapid melanosome aggregation and skin lightening. Somatolactin is also a fish-specific peptide whose expression and secretion are altered in organisms adapted chronically to white/black backgrounds, and that regulates morphological pigmentation. We discuss the evidence for a model whereby in teleosts, MCHL and somatolactin provide the previously unknown link between physiological and morphological pigmentation.

The regulation of skin pigmentation in response to environmental light by pineal Type II opsins and skin melanophore melatonin receptors.

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 Î·m) 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.

Lhx2/9 and Etv1 Transcription Factors have Complementary roles in Regulating the Expression of Guidance Genes slit1 and sema3a.

During neural network development, growing axons read a map of guidance cues expressed in the surrounding tissue that lead the axons toward their targets. In particular, Xenopus retinal ganglion axons use the cues Slit1 and Semaphorin 3a (Sema3a) at a key guidance decision point in the mid-diencephalon in order to continue on to their midbrain target, the optic tectum. The mechanisms that control the expression of these cues, however, are poorly understood. Extrinsic Fibroblast Growth Factor (Fgf) signals are known to help coordinate the development of the brain by regulating gene expression. Here, we propose Lhx2/9 and Etv1 as potential downstream effectors of Fgf signalling to regulate slit1 and sema3a expression in the Xenopus forebrain. We find that lhx2/9 and etv1 mRNAs are expressed complementary to and within slit1/sema3a expression domains, respectively. Our data indicate that Lhx2 functions as an indirect repressor in that lhx2 overexpression within the forebrain downregulates the mRNA expression of both guidance genes, and in vitro lhx2/9 overexpression decreases the activity of slit1 and sema3a promoters. The Lhx2-VP16 constitutive activator fusion reduces sema3a promoter function, and the Lhx2-En constitutive repressor fusion increases slit1 induction. In contrast, etv1 gain of function transactivates both guidance genes in vitro and in the forebrain. Based on these data, together with our previous work, we hypothesize that Fgf signalling promotes both slit1 and sema3a expression in the forebrain through Etv1, while using Lhx2/9 to limit the extent of expression, thereby establishing the proper boundaries of guidance cue expression.

The Expression of Key Guidance Genes at a Forebrain Axon Turning Point Is Maintained by Distinct Fgfr Isoforms but a Common Downstream Signal Transduction Mechanism.

During development the axons of neurons grow toward and locate their synaptic partners to form functional neural circuits. Axons do so by reading a map of guidance cues expressed by surrounding tissues. Guidance cues are expressed at a precise space and time, but how guidance cue expression is regulated, and in a coordinated manner, is poorly understood. Semaphorins (Semas) and Slits are families of molecular ligands that guide axons. We showed previously that fibroblast growth factor (Fgf) signaling maintains sema3a and slit1 forebrain expression in Xenopus laevis, and these two repellents cooperate to guide retinal ganglion cell (RGC) axons away from the mid-diencephalon and on towards the optic tectum. Here, we investigate whether there are common features of the regulatory pathways that control the expression of these two guidance cues at this single axon guidance decision point. We isolated the sema3a proximal promoter and confirmed its responsiveness to Fgf signaling. Through misexpression of truncated Fgf receptors (Fgfrs), we found that sema3a forebrain expression is dependent on Fgfr2-4 but not Fgfr1. This is in contrast to slit1, whose expression we showed previously depends on Fgfr1 but not Fgfr2-4. Using pharmacological inhibitors and misexpression of constitutively active (CA) and dominant negative (DN) signaling intermediates, we find that while distinct Fgfrs regulate these two guidance genes, intracellular signaling downstream of Fgfrs appears to converge along the phosphoinositol 3-kinase (PI3K)-Akt signaling pathway. A common PI3K-Akt signaling pathway may allow for the coordinated expression of guidance cues that cooperate to direct axons at a guidance choice point.

Bifunctional Pyrrolidin-2-one Terminated Manganese Oxide Nanoparticles for Combined Magnetic Resonance and Fluorescence Imaging.

Multimodal probes are an asset for simplified, improved medical imaging. In particular, fluorescence and magnetic resonance imaging (MRI) are sought-after combined capabilities. Here, we show that pyrrolidin-2-one-capped manganese oxide nanoparticles (MnOpyrr NPs) combine MRI with fluorescence microscopy to function as efficient bifunctional bio-nanoprobes. We employ a one-pot synthesis for ca. 10 nm MnO NPs, wherein manganese(II) 2,4-pentadionate is thermally decomposed using pyrrolidin-2-one as a solvent and capping ligand. The MnOpyrr NPs are soluble in water without any further postsynthetic modifications. The r1 relaxivity and r2 /r1 ratio indicate that these NPs are potential T1 MRI contrast agents at clinical (3 T) and ultrahigh (9.4 T) magnetic fields. Serendipitously, the as-prepared NPs are photoluminescent. The unexpected luminescence is ascribed to the modification of the pyrrolidin-2-one during the thermal treatment. MnOpyrr NPs are successfully used to enable fluorescence microscopy of HeLa cells, demonstrating bifunctional imaging capabilities. A low cytotoxic response in two distinct cell types (HeLa, HepG2) supports the suitability of MnOpyrr NPs for biological imaging applications.

Plasticity for colour adaptation in vertebrates explained by the evolution of the genes pomc, pmch and pmchl.

Different camouflages work best with some background matching colour. Our understanding of the evolution of skin colour is based mainly on the genetics of pigmentation ("background matching"), with little known about the evolution of the neuroendocrine systems that facilitate "background adaptation" through colour phenotypic plasticity. To address the latter, we studied the evolution in vertebrates of three genes, pomc, pmch and pmchl, that code for α-MSH and two melanin-concentrating hormones (MCH and MCHL). These hormones induce either dispersion/aggregation or the synthesis of pigments. We find that α-MSH is highly conserved during evolution, as is its role in dispersing/synthesizing pigments. Also conserved is the three-exon pmch gene that encodes MCH, which participates in feeding behaviours. In contrast, pmchl (known previously as pmch), is a teleost-specific intron-less gene. Our data indicate that in zebrafish, pmchl-expressing neurons extend axons to the pituitary, supportive of an MCHL hormonal role, whereas zebrafish and Xenopus pmch+ neurons send axons dorsally in the brain. The evolution of these genes and acquisition of hormonal status for MCHL explain different mechanisms used by vertebrates to background-adapt.

Synthesis and Unprecedented Complexation Properties of ß-Cyclodextrin-Based Ligand for Lanthanide Ions.

Here, we report the synthesis and detailed studies on the coordination chemistry of a novel chemically modified polyaminocarboxylate (5) based on ß-cyclodextrin (CD) scaffold for lanthanides. The target ligand is prepared in a highly efficient manner (seven total steps) from ß-CD using the readily available iminodiacetic acid as a starting material. A propargyl group is attached to the iminodiacetate via N-alkylation, and the obtained derivative is efficiently conjugated to the ß-CD scaffold via the copper(I)-mediated 1,3-dipolar cycloaddition. The generated 1,2,3-triazolmethyl residues advantageously provide a competent chelating group while displacing the metal coordination center away from the primary rim of ß-CD, to afford the required conformational flexibility. The functional groups from each of the two adjacent glucopyranosyl units of ß-CD complete a uniquely created octavalent coordination sphere for lanthanides while still sparing one site for dynamic water coordination. To help study the coordination chemistry of CD ligand 5, we also design a relevant maltoside ligand 6, which faithfully represents one submetal-binding section of ligand 5. Thanks to HRMS and NMR studies, we successfully elucidate the coordination chemistries of synthesized ligands. The octavalent coordination sphere of ligand 5 shows strong binding affinity to lanthanides. By potentiometric titration experiments, ligand 5 is found to bind gadolinium(III), forming 1:1, 1:2, and 1:3 multinuclear complexes with lanthanides, thus possessing great capacity for catalyzing the dynamic water-exchange. Further NMR studies also reveal that the formed ligand 5/Gd(III) complexes show significantly better abilities to alter T1 relaxivities of coordinated water than DOTA-Gd(III) and also some of the synthetic CD probes reported in the literature.

Fibroblast growth factor receptor 1 signaling transcriptionally regulates the axon guidance cue slit1.

Axons sense molecular cues in their environment to arrive at their post-synaptic targets. While many of the molecular cues have been identified, the mechanisms that regulate their spatiotemporal expression remain elusive. We examined here the transcriptional regulation of the guidance gene slit1 both in vitro and in vivo by specific fibroblast growth factor receptors (Fgfrs). We identified an Fgf-responsive 2.3 kb slit1 promoter sequence that recapitulates spatiotemporal endogenous expression in the neural tube and eye of Xenopus embryos. We found that signaling through Fgfr1 is the main regulator of slit1 expression both in vitro in A6 kidney epithelial cells, and in the Xenopus forebrain, even when other Fgfr subtypes are present in cells. These data argue that a specific signaling pathway downstream of Fgfr1 controls in a cell-autonomous manner slit1 forebrain expression and are novel in identifying a specific growth factor receptor for in vivo control of the expression of a key embryonic axon guidance cue.

Seeing the light to change colour: An evolutionary perspective on the role of melanopsin in neuroendocrine circuits regulating light-mediated skin pigmentation.

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.

Interaction and developmental activation of two neuroendocrine systems that regulate light-mediated skin pigmentation.

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.

Two light-activated neuroendocrine circuits arising in the eye trigger physiological and morphological pigmentation.

Two biological processes regulate light-induced skin colour change. A fast 'physiological pigmentation change' (i.e. circadian variations or camouflage) involves alterations in the distribution of pigment containing granules in the cytoplasm of chromatophores, while a slower 'morphological pigmentation change' (i.e. seasonal variations) entails changes in the number of pigment cells or pigment type. Although linked processes, the neuroendocrine coordination triggering each response remains largely obscure. By evaluating both events in Xenopus laevis embryos, we show that morphological pigmentation initiates by inhibiting the activity of the classical retinal ganglion cells. Morphological pigmentation is always accompanied by physiological pigmentation, and a melatonin receptor antagonist prevents both responses. Physiological pigmentation also initiates in the eye, but with repression of melanopsin-expressing retinal ganglion cell activity that leads to secretion of alpha-melanocyte-stimulating hormone (α-MSH). Our findings suggest a model in which eye photoperception links physiological and morphological pigmentation by altering α-MSH and melatonin production, respectively.

Pharmacological induction of skin pigmentation unveils the neuroendocrine circuit regulated by light.

Light-regulated skin colour change is an important physiological process in invertebrates and lower vertebrates, and includes daily circadian variation and camouflage (i.e. background adaptation). The photoactivation of melanopsin-expressing retinal ganglion cells (mRGCs) in the eye initiates an uncharacterized neuroendocrine circuit that regulates melanin dispersion/aggregation through the secretion of alpha-melanocyte-stimulating hormone (α-MSH). We developed experimental models of normal or enucleated Xenopus embryos, as well as in situ cultures of skin of isolated dorsal head and tails, to analyse pharmacological induction of skin pigmentation and α-MSH synthesis. Both processes are triggered by a melanopsin inhibitor, AA92593, as well as chloride channel modulators. The AA9253 effect is eye-dependent, while functional data in vivo point to GABAA receptors expressed on pituitary melanotrope cells as the chloride channel blocker target. Based on the pharmacological data, we suggest a neuroendocrine circuit linking mRGCs with α-MSH secretion, which is used normally during background adaptation.

Melanopsin photoreception in the eye regulates light-induced skin colour changes through the production of α-MSH in the pituitary gland.

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.

Wiring the retinal circuits activated by light during early development.

BACKGROUND: Light 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. RESULTS: We 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. CONCLUSIONS: We 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.

Neuropilin-1 biases dendrite polarization in the retina.

The majority of neurons in the nervous system exhibit a polarized morphology, with multiple short dendrites and a single long axon. It is clear that multiple factors govern polarization in developing neurons, and the biased accumulation of intrinsic determinants to one side of the cell, coupled with responses to asymmetrically localized extrinsic factors, appears to be crucial. A number of intrinsic factors have been identified, but surprisingly little is known about the identity of the extrinsic signals. Here, we show in vivo that neuropilin-1 (Nrp1) and its co-receptor plexinA1 (Plxna1) are necessary to bias the extension of the dendrites of retinal ganglion cells to the apical side of the cell, and ectopically expressed class III semaphorins (Sema3s) disrupt this process. Importantly, the requirement for Nrp1 and Plxna1 in dendrite polarization occurs at a developmental time point after the cells have already extended their basally directed axon. Thus, we propose a novel mechanism whereby an extrinsic factor, probably a Sema3, acts through Nrp1 and Plxna1 to promote the asymmetric outgrowth of dendrites independently of axon polarization.