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.

Plexina4 and cell survival in the developing zebrafish hindbrain.

BACKGROUND: Growth factors are important in the developing and mature nervous system to support the survival of neurons. Developmental signaling molecules are known for their roles in controlling neurogenesis and neural circuit formation. Whether or not these molecules also have roles in cell survival in the developing nervous system is poorly understood. Plexins are a family of transmembrane receptors that bind Semaphorin ligands and are known to function in the guidance of developing axons and blood vessels. RESULTS: In embryonic zebrafish, plexina4 is expressed widely in the brain, becoming largely restricted to the hindbrain as neurogenesis and differentiation proceed. Apoptosis is increased in the embryonic hindbrain of a plexina4ca307/ca307 CRISPR mutant. Based on the literature, we tested the secreted heat shock protein, Clusterin, as a candidate ligand to mediate cell survival through Plexina4. clusterin is expressed by the floor plate of the embryonic zebrafish hindbrain, in proximity to plexina4-expressing hindbrain cells. Morpholino-mediated knockdown of Clusterin increases cell apoptosis in the hindbrain, with additional cell death observed in epistasis experiments where Clusterin is knocked down in a plexina4 mutant background. CONCLUSIONS: Our data suggest that Plexina4 promotes cell survival in the developing zebrafish hindbrain, likely through a pathway independent of Clusterin.

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.

Spatial regulation of amacrine cell genesis by Semaphorin 3f.

PURPOSE: The axonal projections of retinal ganglion cells (RGCs) of the eye are topographically organized so that spatial information from visual images is preserved. This retinotopic organization is established during development by secreted morphogens that pattern domains of transcription factor expression within naso-temporal and dorso-ventral quadrants of the embryonic eye. Poorly understood are the downstream signaling molecules that generate the topographically organized retinal cells and circuits. The secreted signaling molecule Semaphorin 3fa (Sema3fa) belongs to the Sema family of molecules that provide positional information to developing cells. Here, we test a role for Sema3fa in cell genesis of the temporal zebrafish retina. METHODS: We compare retinal cell genesis in wild type and sema3fa CRISPR zebrafish mutants by in situ hybridization and immunohistochemistry. RESULTS: We find that mRNAs for sema3fa and known receptors, neuropilin2b (nrp2b) and plexina1a (plxna1a), are expressed by progenitors of the temporal, but not nasal zebrafish embryonic retina. In the sema3faca304/ca304 embryo, initially the domains of expression for atoh7 and neurod4, transcription factors necessary for the specification of RGCs and amacrine cells, respectively, are disrupted. Yet, post-embryonically only amacrine cells of the temporal retina are reduced in numbers, with both GABAergic and glycinergic subtypes affected. CONCLUSIONS: These data suggest that Sema3fa acts early on embryonic temporal progenitors to control in a spatially-dependent manner the production of amacrine cells, possibly to allow the establishment of neural circuits with domain-specific functions. We propose that spatially restricted extrinsic signals in the neural retina control cell genesis in a domain-dependent manner.

Semaphorin3f as a cardiomyocyte derived regulator of heart chamber development.

BACKGROUND: During development a pool of precursors form a heart with atrial and ventricular chambers that exhibit distinct transcriptional and electrophysiological properties. Normal development of these chambers is essential for full term survival of the fetus, and deviations result in congenital heart defects. The large number of genes that may cause congenital heart defects when mutated, and the genetic variability and penetrance of the ensuing phenotypes, reveals a need to understand the molecular mechanisms that allow for the formation of chamber-specific cardiomyocyte differentiation. METHODS: We used in situ hybridization, immunohistochemistry and functional analyses to identify the consequences of the loss of the secreted semaphorin, Sema3fb, in the development of the zebrafish heart by using two sema3fb CRISPR mutant alleles. RESULTS: We find that in the developing zebrafish heart sema3fb mRNA is expressed by all cardiomyocytes, whereas mRNA for a known receptor Plexina3 (Plxna3) is expressed preferentially by ventricular cardiomyocytes. In sema3fb CRISPR zebrafish mutants, heart chamber development is impaired; the atria and ventricles of mutants are smaller in size than their wild type siblings, apparently because of differences in cell size and not cell numbers. Analysis of chamber differentiation indicates defects in chamber specific gene expression at the border between the ventricular and atrial chambers, with spillage of ventricular chamber genes into the atrium, and vice versa, and a failure to restrict specialized cardiomyocyte markers to the atrioventricular canal (AVC). The hypoplastic heart chambers are associated with decreased cardiac output and heart edema. CONCLUSIONS: Based on our data we propose a model whereby cardiomyocytes secrete a Sema cue that, because of spatially restricted expression of the receptor, signals in a ventricular chamber-specific manner to establish a distinct border between atrial and ventricular chambers that is important to produce a fully functional heart. Video abstract.

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.

Endothelial Semaphorin 3fb regulates Vegf pathway-mediated angiogenic sprouting.

Vessel growth integrates diverse extrinsic signals with intrinsic signaling cascades to coordinate cell migration and sprouting morphogenesis. The pro-angiogenic effects of Vascular Endothelial Growth Factor (VEGF) are carefully controlled during sprouting to generate an efficiently patterned vascular network. We identify crosstalk between VEGF signaling and that of the secreted ligand Semaphorin 3fb (Sema3fb), one of two zebrafish paralogs of mammalian Sema3F. The sema3fb gene is expressed by endothelial cells in actively sprouting vessels. Loss of sema3fb results in abnormally wide and stunted intersegmental vessel artery sprouts. Although the sprouts initiate at the correct developmental time, they have a reduced migration speed. These sprouts have persistent filopodia and abnormally spaced nuclei suggesting dysregulated control of actin assembly. sema3fb mutants show simultaneously higher expression of pro-angiogenic (VEGF receptor 2 (vegfr2) and delta-like 4 (dll4)) and anti-angiogenic (soluble VEGF receptor 1 (svegfr1)/ soluble Fms Related Receptor Tyrosine Kinase 1 (sflt1)) pathway components. We show increased phospho-ERK staining in migrating angioblasts, consistent with enhanced Vegf activity. Reducing Vegfr2 kinase activity in sema3fb mutants rescues angiogenic sprouting. Our data suggest that Sema3fb plays a critical role in promoting endothelial sprouting through modulating the VEGF signaling pathway, acting as an autocrine cue that modulates intrinsic growth factor signaling.

Retinal Pigment Epithelium and Neural Retinal Progenitors Interact via Semaphorin 6D to Facilitate Optic Cup Morphogenesis.

Cell movement propels embryonic tissues to acquire shapes required for mature function. The movements are driven both by acto-myosin signaling and by cells interacting with the extracellular matrix (ECM). Unknown is whether cell-cell interactions within a tissue are also required, and the molecular mechanisms by which such communication might occur. Here, we use the developing visual system of zebrafish as a model to understand the role cell-cell communication plays in tissue morphogenesis in the embryonic nervous system. We identify that cell-cell-mediated contact between two distinct cell populations, progenitors of the neural retina and retinal pigment epithelium (RPE), facilitates epithelial flow to produce the mature cupped retina. We identify for the first time the need in eye morphogenesis for distinct populations of progenitors to interact, and suggest a novel role for a member of a key developmental signaling family, the transmembrane Semaphorin6d, as mediating communication between distinct cell types to control tissue morphogenesis.

Semaphorin 3fa Controls Ocular Vascularization From the Embryo Through to the Adult.

Purpose: Pathological blood vessel growth in the eye is implicated in several diseases that result in vision loss, including age-related macular degeneration and diabetic retinopathy. The limits of current disease therapies have created the need to identify and characterize new antiangiogenic drugs. Here, we identify the secreted chemorepellent semaphorin-3fa (Sema3fa) as an endogenous anti-angiogenic in the eye. Methods: We generated a CRISPR/Cas9 sema3fa zebrafish mutant line, sema3faca304/304. We assessed the retinal and choroidal vasculature in both larval and adult wild-type and sema3fa mutant zebrafish. Results: We find sema3fa mRNA is expressed by the ciliary marginal zone, neural retina, and retinal pigment epithelium of zebrafish larvae as choroidal vascularization emerges and the hyaloid/retinal vasculature is remodeled. The hyaloid vessels of sema3fa mutants develop appropriately but fail to remodel during the larval period, with adult mutants exhibiting a denser network of capillaries in the retinal periphery than seen in wild-type. The choroid vasculature is also defective in that it develops precociously, and aberrant, leaky sprouts are present in the normally avascular outer retina of both sema3faca304/304 larvae and adult fish. Conclusions: Sema3fa is a key endogenous signal for maintaining an avascular retina and preventing pathologic vascularization. Furthermore, we provide a new experimentally accessible model for studying choroid neovascularization (CNV) resulting from primary changes in the retinal environment that lead to downstream vessel infiltration.

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.

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.

Polarity and morphogenesis of the eye epithelium requires the adhesion junction associated adaptor protein Traf4.

During development, neuroepithelial progenitors acquire apico-basal polarity and adhere to one another via apically located tight and adherens junction complexes. This polarized neuroepithelium must continue to integrate cells arising through cell divisions and intercalation, and allow for cell movements, at the same time as undergoing morphogenesis. Cell proliferation, migration and intercalation all occur in the morphing embryonic eye. To understand how eye development might depend on dynamic epithelial adhesion, we investigated the function of a known regulator of junctional plasticity, Tumour necrosis factor receptor-associated factor 4 (Traf4). traf4a mRNA is expressed in the developing eye vesicle over the period of optic cup morphogenesis, and Traf4a loss leads to disrupted evagination and elongation of the eye vesicles, and aberrant organization and apico-basal polarity of the eye epithelium. We propose a model whereby Traf4a regulates apical junction plasticity in nascent eye epithelium, allowing for its polarization and morphogenesis. Symbols and Abbreviations: AB: apico-basal; aPKC: atypical protein kinase-C; CRISPR: clustered regularly-interspaced short palindromic repeats; GFP: green fluorescent protein; hpf: hours post-fertilization; MO: antisense morpholino oligonucleotide; pHH3: phospho histone H3; ss: somite stage; Traf4: Tumour necrosis factor receptor-associated factor 4; ZO-1: zona occludens-1.

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.

Podiatry intervention versus usual care to prevent falls in care homes: pilot randomised controlled trial (the PIRFECT study).

BACKGROUND: Common foot problems are independent risk factors for falls in older people. There is evidence that podiatry can prevent falls in community-dwelling populations. The feasibility of implementing a podiatry intervention and trial in the care home population is unknown. To inform a potential future definitive trial, we performed a pilot randomised controlled trial to assess: (i) the feasibility of a trial of a podiatry intervention to reduce care home falls, and (ii) the potential direction and magnitude of the effect of the intervention in terms of number of falls in care home residents. METHODS: Informed by Medical Research Council guidance on developing and evaluating complex interventions, we conducted a single blind, pilot randomised controlled trial in six care homes in the East of Scotland. Participants were randomised to either: (i) a three month podiatry intervention comprising core podiatry care, foot and ankle exercises, orthoses and footwear provision or (ii) usual care. Falls-related outcomes (number of falls, time to first fall) and feasibility-related outcomes (recruitment, retention, adherence, data collection rates) were collected. Secondary outcomes included: generic health status, balance, mobility, falls efficacy, and ankle joint strength. RESULTS: 474 care home residents were screened. 43 (9.1%) participants were recruited: 23 to the intervention, 20 to control. Nine (21%) participants were lost to follow-up due to declining health or death. It was feasible to deliver the trial elements in the care home setting. 35% of participants completed the exercise programme. 48% reported using the orthoses 'all or most of the time'. Completion rates of the outcome measures were between 93% and 100%. No adverse events were reported. At the nine month follow-up period, the intervention group per-person fall rate was 0.77 falls vs. 0.83 falls in the control group. CONCLUSIONS: A podiatry intervention to reduce falls can be delivered to care home residents within a pilot randomised controlled trial of the intervention. Although not powered to determine effectiveness, these preliminary data provide justification for a larger trial, incorporating a full process evaluation, to determine whether this intervention can significantly reduce falls in this high-risk population. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02178527 ; Date of registration: 17 June 2014.