Microglia are resident immune cells in the central nervous system, including the retina that surveil the environment for damage and infection. Following retinal damage, microglia undergo morphological changes, migrate to the site of damage, and express and secrete pro-inflammatory signals. In the zebrafish retina, inflammation induces the reprogramming and proliferation of Müller glia and the regeneration of neurons following damage or injury. Immunosuppression or pharmacological ablation of microglia reduce or abolish Müller glia proliferation. We evaluated the retinal architecture and retinal regeneration in adult zebrafish irf8 mutants, which have significantly depleted numbers of microglia. We show that irf8 mutants have normal retinal structure at 3 months post fertilization (mpf) and 6 mpf but fewer cone photoreceptors by 10 mpf. Surprisingly, light-induced photoreceptor ablation induced Müller glia proliferation in irf8 mutants and cone and rod photoreceptor regeneration. Light-damaged retinas from both wild-type and irf8 mutants show upregulated expression of mmp-9, il8, and tnfβ pro-inflammatory cytokines. Our data demonstrate that adult zebrafish irf8 mutants can regenerate normally following acute retinal injury. These findings suggest that microglia may not be essential for retinal regeneration in zebrafish and that other mechanisms can compensate for the reduction in microglia numbers.

Emerging evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR), preceding the development of microvascular abnormalities. Here, we assessed the impact of neuroinflammation on the retina of diabetic-induced rats. For this aim we have used a two-photon microscope to image the photoreceptors (PRs) at different eccentricities in unstained retinas obtained from both control (N = 4) and pathological rats (N = 4). This technique provides high-resolution images where individual PRs can be identified. Within each image, every PR was located, and its transversal area was measured and used as an objective parameter of neuroinflammation. In control samples, the size of the PRs hardly changed with retinal eccentricity. On the opposite end, diabetic retinas presented larger PR transversal sections. The ratio of PRs suffering from neuroinflammation was not uniform across the retina. Moreover, the maximum anatomical resolving power (in cycles/deg) was also calculated. This presents a double-slope pattern (from the central retina towards the periphery) in both types of specimens, although the values for diabetic retinas were significantly lower across all retinal locations. The results show that chronic retinal inflammation due to diabetes leads to an increase in PR transversal size. These changes are not uniform and depend on the retinal location. Two-photon microscopy is a useful tool to accurately characterize and quantify PR inflammatory processes and retinal alterations.

Root system architecture (RSA) encompasses a range of physical root attributes, including the lateral roots (LRs), root hairs and adventitious roots, in addition to the primary or main root. This overall structure is a crucial trait for efficient water and mineral capture alongside providing anchorage to the plant in the soil and is vital for plant productivity and fitness. RSA dynamics are dependent upon various environmental cues such as light, soil pH, water, mineral nutrition and the belowground microbiome. Among these factors, light signaling through HY5 significantly influences the flexibility of RSA by controlling different signaling pathways that converge at photoreceptors-mediated signaling, also present in the \'hidden half\'. Furthermore, several phytohormones also drive the formation and emergence of LRs and are critical to harmonize intra and extracellular stimuli in this regard. This review endeavors to elucidate the impact of these interactions on RSA, with particular emphasis on LR development and to enhance our understanding of the fundamental mechanisms governing the light-regulation of LR growth and physiology.

Exposure to the non-protein amino acid cyanotoxin β-N-methylamino-L-alanine (BMAA), released by cyanobacteria found in many water reservoirs has been associated with neurodegenerative diseases. We previously demonstrated that BMAA induced cell death in both retina photoreceptors (PHRs) and amacrine neurons by triggering different molecular pathways, as activation of NMDA receptors and formation of carbamate-adducts was only observed in amacrine cell death. We established that activation of Retinoid X Receptors (RXR) protects retinal cells, including retina pigment epithelial (RPE) cells from oxidative stress-induced apoptosis. We now investigated the mechanisms underlying BMAA toxicity in these cells and those involved in RXR protection. BMAA addition to rat retinal neurons during early development in vitro increased reactive oxygen species (ROS) generation and polyADP ribose polymers (PAR) formation, while pre-treatment with serine (Ser) before BMAA addition decreased PHR death. Notably, RXR activation with the HX630 agonist prevented BMAA-induced death in both neuronal types, reducing ROS generation, preserving mitochondrial potential, and decreasing TUNEL-positive cells and PAR formation. This suggests that BMAA promoted PHR death by substituting Ser in polypeptide chains and by inducing polyADP ribose polymerase activation. BMAA induced cell death in ARPE-19 cells, a human epithelial cell line; RXR activation prevented this death, decreasing ROS generation and caspase 3/7 activity. These findings suggest that RXR activation prevents BMAA harmful effects on retinal neurons and RPE cells, supporting this activation as a broad-spectrum strategy for treating retina degenerations.

UNASSIGNED: An animal\'s ability to discriminate between differing wavelengths of light (i.e., color vision) is mediated, in part, by a subset of photoreceptor cells that express opsins with distinct absorption spectra. In Drosophila R7 photoreceptors, expression of the rhodopsin molecules, Rh3 or Rh4, is determined by a stochastic process mediated by the transcription factor spineless. The goal of this study was to identify additional factors that regulate R7 cell fate and opsin choice using a Genome Wide Association Study (GWAS) paired with transcriptome analysis via RNA-Seq.
UNASSIGNED: We examined Rh3 and Rh4 expression in a subset of fully-sequenced inbred strains from the Drosophila Genetic Reference Panel and performed a GWAS to identify 42 naturally-occurring polymorphisms-in proximity to 28 candidate genes-that significantly influence R7 opsin expression. Network analysis revealed multiple potential interactions between the associated candidate genes, spineless and its partners. GWAS candidates were further validated in a secondary RNAi screen which identified 12 lines that significantly reduce the proportion of Rh3 expressing R7 photoreceptors. Finally, using RNA-Seq, we demonstrated that all but four of the GWAS candidates are expressed in the pupal retina at a critical developmental time point and that five are among the 917 differentially expressed genes in sevenless mutants, which lack R7 cells.
UNASSIGNED: Collectively, these results suggest that the relatively simple, binary cell fate decision underlying R7 opsin expression is modulated by a larger, more complex network of regulatory factors. Of particular interest are a subset of candidate genes with previously characterized neuronal functions including neurogenesis, neurodegeneration, photoreceptor development, axon growth and guidance, synaptogenesis, and synaptic function.

Non-confocal split-detection imaging reveals the cone photoreceptor inner segment mosaic in a plethora of retinal conditions, with the potential of providing insight to ageing, disease, and response to treatment processes, in vivo, and allows the screening of candidates for cell rescue therapies. This imaging modality complements confocal reflectance adaptive optics scanning light ophthalmoscopy, which relies on the waveguiding properties of cones, as well as their orientation toward the pupil. Split-detection contrast, however, is directional, with each cone inner segment appearing as opposite dark and bright semicircles, presenting a challenge for either manual or automated cell identification. Quadrant-detection imaging, an evolution of split detection, could be used to generate images without directional dependence. Here, we demonstrate how the embossed-filtered quadrant-detection images, originally proposed by Migacz et al. for visualising hyalocytes, can also be used to generate photoreceptor mosaic images with better and non-directional contrast for improved visualisation. As a surrogate of visualisation improvement between legacy split-detection images and the images resulting from the method described herein, we provide preliminary results of simple image processing routines that may enable the automated identification of generic image features, as opposed to complex algorithms developed specifically for photoreceptor identification, in pathological retinas.

Unlike humans, teleosts like zebrafish exhibit robust retinal regeneration after injury from endogenous stem cells. However, it is unclear if regenerating cone photoreceptors regain physiological function and integrate correctly into post-synaptic circuits. We used two-photon calcium imaging of living adult retina to examine photoreceptor responses before and after light-induced lesions. To assess functional recovery of cones and downstream outer retinal circuits, we exploited color opponency; UV cones exhibit intrinsic Off-response to blue light, but On-response to green light, which depends on feedback signals from outer retinal circuits. Accordingly, we assessed the presence and quality of Off- vs. On-responses and found that regenerated UV cones regain both Off-responses to short-wavelength and On-responses to long-wavelength light within 3 months after lesion. Therefore, physiological circuit functionality is restored in regenerated cone photoreceptors, suggesting that inducing endogenous regeneration is a promising strategy for human retinal repair.

Stargardt disease (STGD1), associated with biallelic variants in the ABCA4 gene, is the most common heritable macular dystrophy and is currently untreatable. To identify potential treatment targets, we characterized surviving STGD1 photoreceptors. We used clinical data to identify macular regions with surviving STGD1 photoreceptors. We compared the hyperreflective bands in the optical coherence tomographic (OCT) images that correspond to structures in the STGD1 photoreceptor inner segments to those in controls. We used adaptive optics scanning light ophthalmoscopy (AO-SLO) to study the distribution of cones and AO-OCT to evaluate the interface of photoreceptors and retinal pigment epithelium (RPE). We found that the profile of the hyperreflective bands differed dramatically between patients with STGD1 and controls. AO-SLOs showed patches in which cone densities were similar to those in healthy retinas and others in which the cone population was sparse. In regions replete with cones, there was no debris at the photoreceptor-RPE interface. In regions with sparse cones, there was abundant debris. Our results raise the possibility that pharmaceutical means may protect surviving photoreceptors and so mitigate vision loss in patients with STGD1.

Primary cilia are sensory organelles that extend from the cellular membrane and are found in a wide range of cell types. Cilia possess a plethora of vital components that enable the detection and transmission of several signaling pathways, including Wnt and Shh. In turn, the regulation of ciliogenesis and cilium length is influenced by various factors, including autophagy, organization of the actin cytoskeleton, and signaling inside the cilium. Irregularities in the development, maintenance, and function of this cellular component lead to a range of clinical manifestations known as ciliopathies. The majority of people with ciliopathies have a high prevalence of retinal degeneration. The most common theory is that retinal degeneration is primarily caused by functional and developmental problems within retinal photoreceptors. The contribution of other ciliated retinal cell types to retinal degeneration has not been explored to date. In this review, we examine the occurrence of primary cilia in various retinal cell types and their significance in pathology. Additionally, we explore potential therapeutic approaches targeting ciliopathies. By engaging in this endeavor, we present new ideas that elucidate innovative concepts for the future investigation and treatment of retinal ciliopathies.

The distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and for generating visual pigments for photoreceptor cell function. ROL circulates in the blood bound to the retinol binding protein 4 (RBP4) as RBP4-ROL. Two membrane receptors, RBPR2 in the liver and STRA6 in the eye are proposed to bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Here, we present a longitudinal investigation towards the importance of RBPR2 and influence of the diet on systemic retinoid homeostasis for visual function. Age matched Rbpr2-KO (Rbpr2 -/- ) and wild-type (WT) mice were fed either a vitamin A sufficient (VAS) or a vitamin A deficient (VAD) diet. At 3- and 6-months, we performed retinoid quantification of ocular and non-ocular tissues using HPLC analysis and complemented the data with visual physiology, rhodopsin quantification by spectrophotometry, and biochemical analysis. At 3-months and compared to WT mice, Rbpr2 -/- mice fed either vitamin A diets displayed lower scotopic and photopic electroretinogram (ERG) responses, which correlated with HPLC analysis that revealed Rbpr2 -/- mice had significantly lower hepatic and ocular retinoid content. Interestingly, with the exception of the liver, long-term feeding of Rbpr2 -/- mice with a VAS diet promoted all-trans retinol accumulation in most peripheral tissues. However, even under VAS dietary conditions significant amounts of unliganded opsins in rods, together with decreased visual responses were evident in aged mice lacking RBPR2, when compared to WT mice. Together, our analyses characterize the molecular events underlying nutritional blindness in a novel mouse model and indicate that loss of the liver specific RBP4-ROL receptor, RBPR2, influences systemic retinoid homeostasis and rhodopsin synthesis, which causes profound visual function defects under severe vitamin A deficiency conditions.