Vertebrate Retina Research Articles

In Vivo Imaging of Retinal and Choroidal Morphology and Vascular Plexuses of Vertebrates Using Swept-Source Optical Coherence Tomography.

PurposeTo perform in vivo evaluation of the structural morphology and vascular plexuses of the neurosensory retina and choroid across vertebrate species using swept-source optical coherence tomography (SS-OCT) and SS-OCT angiography (SS-OCTA) imaging.MethodsA custom-built SS-OCT system with an incorporated flexible imaging arm was used to acquire the three-dimensional (3D) retinal OCT and vascular OCTA data of five different vertebrates: a mouse (C57BL/6J), a rat (Long Evans), a gray short-tailed opossum (Monodelphis domestica), a white sturgeon (Acipenser transmontanus), and a great horned owl (Bubo virginianus).ResultsIn vivo structural morphology of the retina and choroid, as well as en face OCTA images of retinal and choroidal vasculature of all species were generated. The retinal morphology and vascular plexuses were similar between rat and mouse, whereas distinct choroidal and paired superficial vessels were observed in the opossum retina. The retinal and vascular structure of the sturgeon, as well as the pecten oculi and overlying the avascular and choroidal vasculature in the owl retina are reported in vivo.ConclusionsA high-quality two-dimensional and 3D in vivo visualization of the retinal structures and en face visualization of the retina and choroidal vascular plexus of vertebrates was possible. Our studies affirm that SS-OCT and SS-OCTA are viable methods for evaluating the in vivo retinal and choroidal structure across terrestrial, aquatic, and aerial vertebrates.Translational RelevanceIn vivo characterization of retinal morphology and vasculature plexus of multiple species using SS-OCT and SS-OCTA imaging can increase the pool of species available as models of human retinal diseases.

Specialization of the photoreceptor transcriptome by Srrm3-dependent microexons is required for outer segment maintenance and vision

Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport. This microexon program is regulated by Srrm3, a paralog of the neural microexon regulator Srrm4. Despite the fact that both proteins positively regulate retina microexons invitro, only Srrm3 is highly expressed in mature photoreceptors. Its deletion in zebrafish results in widespread down-regulation of microexon inclusion from early developmental stages, followed by other transcriptomic alterations, severe photoreceptor defects, and blindness. These results shed light on the transcriptomic specialization and functionality of photoreceptors, uncovering unique cell type-specific roles for Srrm3 and microexons with implications for retinal diseases.

Self-Organization of the Retina during Eye Development, Retinal Regeneration In Vivo, and in Retinal 3D Organoids In Vitro.

Self-organization is a process that ensures histogenesis of the eye retina. This highly intricate phenomenon is not sufficiently studied due to its biological complexity and genetic heterogeneity. The review aims to summarize the existing central theories and ideas for a better understanding of retinal self-organization, as well as to address various practical problems of retinal biomedicine. The phenomenon of self-organization is discussed in the spatiotemporal context and illustrated by key findings during vertebrate retina development in vivo and retinal regeneration in amphibians in situ. Described also are histotypic 3D structures obtained from the disaggregated retinal progenitor cells of birds and retinal 3D organoids derived from the mouse and human pluripotent stem cells. The review highlights integral parts of retinal development in these conditions. On the cellular level, these include competence, differentiation, proliferation, apoptosis, cooperative movements, and migration. On the physical level, the focus is on the mechanical properties of cell- and cell layer-derived forces and on the molecular level on factors responsible for gene regulation, such as transcription factors, signaling molecules, and epigenetic changes. Finally, the self-organization phenomenon is discussed as a basis for the production of retinal organoids, a promising model for a wide range of basic scientific and medical applications.

Timing and Distribution of Mitotic Activity in the Retina During Precocial and Altricial Modes of Avian Development

During development of the vertebrate retina, mitotic activity is defined as apical when is located at the external surface of the neuroepithelium or as non-apical when is found in more internal regions. Apical mitoses give rise to all retinal cell types. Non-apical mitoses are linked to committed horizontal cell precursors that subsequently migrate vitreo-sclerally, reaching their final position in the outer surface of the inner nuclear layer, where they differentiate. Previous studies have suggested differences in the timing of retinal maturation between altricial and precocial bird species. In the present study we analyze qualitatively and quantitatively the mitotic activity in the developing retina of an altricial (zebra finch, Taeniopygia guttata) and a precocial (Japanese quail, Coturnix coturnix) bird species. We found that pHisH3-immunoreactive apical and non-apical mitoses were abundant in the T. guttata retina at the hatching stage. In contrast, pHisH3 immunoreactivity almost disappeared from the quail retina at the embryonic day 10 (E10). Furthermore, we also found that the onset of the appearance of non-apical mitoses occurred at later stages in the altricial bird species than in the precocial one. The disappearance of apical mitoses and the spatiotemporal distribution of non-apical mitoses followed central to peripheral and dorsal to ventral gradients, similar to gradients of cell differentiation described in the retina of birds. Therefore, these results suggest that retinal neurogenesis is active at the hatching stage in T. guttata, and that horizontal cell differentiation is delayed in the altricial bird species compared to the precocial one. Together, this study reveals important insights into the timing differences that regulate bird retinal maturation and provides a better understanding of the evolution of avian altriciality and precociality.

Retinal pigment epithelium 65 kDa protein (RPE65): An update

Vertebrate vision critically depends on an 11-cis-retinoid renewal system known as the visual cycle. At the heart of this metabolic pathway is an enzyme known as retinal pigment epithelium 65kDa protein (RPE65), which catalyzes an unusual, possibly biochemically unique, reaction consisting of a coupled all-trans-retinyl ester hydrolysis and alkene geometric isomerization to produce 11-cis-retinol. Early work on this isomerohydrolase demonstrated its membership to the carotenoid cleavage dioxygenase superfamily and its essentiality for 11-cis-retinal production in the vertebrate retina. Three independent studies published in 2005 established RPE65 as the actual isomerohydrolase instead of a retinoid-binding protein as previously believed. Since the last devoted review of RPE65 enzymology appeared in this journal, major advances have been made in a number of areas including our understanding of the mechanistic details of RPE65 isomerohydrolase activity, its phylogenetic origins, the relationship of its membrane binding affinity to its catalytic activity, its role in visual chromophore production for rods and cones, its modulation by macromolecules and small molecules, and the involvement of RPE65 mutations in the development of retinal diseases. In this article, I will review these areas of progress with the goal of integrating results from the varied experimental approaches to provide a comprehensive picture of RPE65 biochemistry. Key outstanding questions that may prove to be fruitful future research pursuits will also be highlighted.

Elucidating the Role of the Electron Transport Chain in Retinal Progenitor Cells and Neuro‐ regeneration

In the retinas of non‐mammalian vertebrates, such as zebrafish, neural regeneration is mediated by the molecular reprogramming of MGs into progenitor‐like cells. While mammalian MGs do not exhibit the regenerative potential of the zebrafish, it has been shown that MGs of mammals display limited‐ and transient‐entry into the cell cycle in response to damage. Therefore, an intrinsic proliferative and/or regenerative block likely prevents mammalian MGs from undergoing sustained cell cycle re‐entry and acquiring a multipotential retinal progenitor‐like state. Using a gain‐of‐function strategy we bypassed the Hippo signaling pathway and induced expression of a mutant version of YAP (YAP5SA) in MGs. Lineage tracing and histological analyses of these YAP5SA‐mutant retinas showed that MGs that expressed YAP5SA entered the cell cycle and formed radial clusters of proliferative cells strikingly similar to clonally expanding MG‐derived progenitors of the regenerating zebrafish retina. This finding indicated that YAP5SA is capable of spontaneously driving MGs into the cell cycle. Whether this YAP‐mediated reprogramming of MGs is sufficient to induce differentiation of MGs into fully functional neurons is undetermined. Subsequent single cell RNA sequencing analysis of the YAP5SA‐mutant retinas revealed that the proliferative event of YAP5SA‐positive cells occurred coincident with a dramatic downregulation of genes required for mitochondrial metabolic processes indicating that a metabolic shift is part of the MG reprogramming event. In a variety of cellular contexts, mitochondrial function and the electron transport chain (ETC) activity have emerged as integral regulators of the cell cycle and cell fate. The factors regulating a mitochondrial metabolic reprogramming and cell cycle are coupled but unknown. We generated a mouse line to conditionally knock out (CKO) the mitochondrial transcription factor A (Tfam) to target the ETC. In the CKO developing retinas, loss of Tfam results in loss of ETC activity. Preliminary histological analysis of CKO developing retinas show that loss of Tfam delays progenitor cell cycle exit and reduces neurogenesis resulting in a hypoplastic adult retina. Our findings so far indicate that unless retinal progenitor cells have a functional ETC‐driven oxidative activity, they remain within the cell cycle compromising the normal neural developmental program. The overall goal of this studies will be to determine how the RPC cell cycle machinery interfaces with ETC to drive cell cycle exit and neural differentiation. A better understanding of these requirements will have broad implications for the entire field of neural regeneration.

Photoreceptor physiology and evolution: cellular and molecular basis of rod and cone phototransduction.

The detection of light in the vertebrate retina utilizes a duplex system of closely related rod and cone photoreceptors: cones respond extremely rapidly, and operate at 'photopic' levels of illumination, from moonlight upwards; rods respond much more slowly, thereby obtaining greater sensitivity, and function effectively only at 'scotopic' levels of moonlight and lower. Rods and cones employ distinct isoforms of many of the proteins in the phototransduction cascade, and they thereby represent a unique evolutionary system, whereby the same process (the detection of light) uses a distinct set of genes in two classes of cell. The molecular mechanisms of phototransduction activation are described, and the classical quantitative predictions for the onset phase of the electrical response to light are developed. Recent work predicting the recovery phase of the rod's response to intense flashes is then presented, that provides an accurate account of the time that the response spends in saturation. Importantly, this also provides a new estimate for the rate at which a single rhodopsin activates molecules of the G-protein, transducin, that is substantially higher than other estimates in the literature. Finally, the evolutionary origin of the phototransduction proteins in rods and cones is examined, and it is shown that most of the rod/cone differences were established at the first of the two rounds of whole-genome duplication more than 500 million years ago.

Compensatory engulfment and Müller glia reactivity in the absence of microglia.

Microglia are known for important phagocytic functions in the vertebrate retina. Reports also suggest that Müller glia have phagocytic capacity, though the relative levels and contexts in which this occurs remain to be thoroughly examined. Here, we investigate Müller glial engulfment of dying cells in the developing zebrafish retina in the presence and absence of microglia, using a genetic mutant in which microglia do not develop. We show that in normal conditions clearance of dying cells is dominated by microglia; however, Müller glia do have a limited clearance role. In retinas lacking intact microglial populations, we found a striking increase in the engulfment load assumed by the Müller glia, which displayed prominent cellular compartments containing apoptotic cells, several of which localized with the early phagosome/endosome marker Rab5. Consistent with increased engulfment, lysosomal staining was also increased in Müller glia in the absence of microglia. Increased engulfment load led to evidence of Müller glia reactivity including upregulation of gfap but did not trigger cell cycle re-entry by differentiated Müller glia. Our work provides important insight into the phagocytic capacity of Müller glia and the ability for compensatory functions and downstream effects. Therefore, effects of microglial deficiency or depletion on other glial cell types should be well-considered in experimental manipulations, in neurodegenerative disease, and in therapeutic approaches that target microglia. Our findings further justify future work to understand differential mechanisms and contexts of phagocytosis by glial cells in the central nervous system, and the significance of these mechanisms in health and disease.

Mitochondrial ATP synthesis activated by exciton energy transfer from Müller cell intermediate filaments

• Exciton energy transferred from IFs to mitochondria accelerated ATP production rate. • ATP production rate explored in function of mitochondrial concentration. • ATP production rate explored in function of intensity of light that produced excitons. ATP production by mitochondria isolated from yeast cells ( S. cerevisiae ) was accelerated by exciton energy transfer from intermediate filaments (IFs). The effect was dependent on mitochondrial concentration and the intensity of light used to generate excitons. Presumably, mitochondrial cytochrome C (CC) was activated by IF exciton energy transfer to CC in the inner mitochondrial membrane (IMM), generating electronically excited CC, and the respective excitation energy was then used in the ATP production chain. A qualitative model was proposed describing observations, although more experiments are required before the detailed mechanisms could be deduced. The present results support the earlier proposed hypothesis of ATP production in vertebrate retina by excess photons in daytime, when retina needs extra energy for recovering the used opsins. Additionally, it shows that at early stages of evolution mitochondria could have been using solar radiation to produce ATP.

Vision and retina evolution: How to develop a retina.

Early in vertebrate evolution, a single homeobox (Hox) cluster in basal chordates was quadrupled to generate the Hox gene clusters present in extant vertebrates. Here we ask how this expanded gene pool may have influenced the evolution of the visual system. We suggest that a single neurosensory cell type split into ciliated sensory cells (photoreceptors, which transduce light) and retinal ganglion cells (RGC, which project to the brain). In vertebrates, development of photoreceptors is regulated by the basic helix-loop-helix (bHLH) transcription factor Neurod1 whereas RGC development depends on Atoh7 and related bHLH genes. Lancelet (a basal chordate) does not express Neurod or Atoh7 and possesses a few neurosensory cells with cilia that reach out of the opening of the neural tube. Sea-squirts (Ascidians) do not express Neurod and express a different bHLH gene, Atoh8, that is likely expressed in the anterior vesicle. Recent data indicate the neurosensory cells in lancelets may correspond to three distinct eye fields in ascidians, which in turn may be the basis of the vertebrate retina, pineal and parapineal. In this review we contrast the genetic control of visual structure development in these chordates with that of basal vertebrates such as lampreys and hagfish, and jawed vertebrates. We propose an evolutionary sequence linking whole-genome duplications, initially to a split between photoreceptor and projection neurons (RGC) and subsequently between pineal and lateral eye structures.

A 3D molecular map of the cavefish neural plate illuminates eye-field organization and its borders in vertebrates.

The vertebrate retinas originate from a specific anlage in the anterior neural plate called the eye field. Its identity is conferred by a set of 'eye transcription factors', whose combinatorial expression has been overlooked. Here, we use the dimorphic teleost Astyanax mexicanus, which develops proper eyes in the wild type and smaller colobomatous eyes in the blind cavefish embryos, to unravel the molecular anatomy of the eye field and its variations within a species. Using a series of markers (rx3, pax6a, cxcr4b, zic1, lhx2, emx3 and nkx2.1a), we draw a comparative 3D expression map at the end of gastrulation/onset of neurulation, which highlights hyper-regionalization of the eye field into sub-territories of distinct sizes, shapes, cell identities and combinatorial gene expression levels along the three body axes. All these features show significant variations in the cavefish natural mutant. We also discover sub-domains within the prospective telencephalon and characterize cell identities at the frontiers of the eye field. We propose putative fates for some of the characterized eye-field subdivisions, and suggest the existence of a trade-off between some subdivisions in the two Astyanax morphs on a micro-evolutionary scale.

Probing Photoreceptor Outer Segment Phagocytosis by the RPE In Vivo: Models and Methodologies.

In the vertebrate retina, the light-sensitive photoreceptor rods and cones constantly undergo renewal by generating new portions of the outer segment and shedding their distal, spent tips. The neighboring RPE provides the critical function of engulfing the spent material by phagocytosis. RPE phagocytosis of shed rod outer segment fragments is a circadian process that occurs in a burst of activity shortly after daily light onset with low activity at other times, a rhythm that has been reported for many species and over 50 years. In this review, we compare studies on the rhythm and quantity of RPE phagocytosis using different in vivo model systems and assessment methods. We discuss how measurement methodology impacts the observation and analysis of RPE phagocytosis. Published studies on RPE phagocytosis investigating mice further suggest that differences in genetic background and housing conditions may affect results. Altogether, a comparison between RPE phagocytosis studies performed using differing methodology and strains of the same species is not as straightforward as previously thought.

Structural and functional analysis of the human cone-rod homeobox transcription factor.

The cone‐rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone‐rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 25 years since its initial characterization, there are no high‐resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small‐angle X‐ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX‐HD). We find that the CRX‐HD is a compact, globular monomer in solution that can specifically bind functional cis‐regulatory elements encoded upstream of retina‐specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease‐causing mutant protein variants.

Strip1 regulates retinal ganglion cell survival by suppressing Jun-mediated apoptosis to promote retinal neural circuit formation.

In the vertebrate retina, an interplay between retinal ganglion cells (RGCs), amacrine (AC), and bipolar (BP) cells establishes a synaptic layer called the inner plexiform layer (IPL). This circuit conveys signals from photoreceptors to visual centers in the brain. However, the molecular mechanisms involved in its development remain poorly understood. Striatin-interacting protein 1 (Strip1) is a core component of the striatin-interacting phosphatases and kinases (STRIPAK) complex, and it has shown emerging roles in embryonic morphogenesis. Here, we uncover the importance of Strip1 in inner retina development. Using zebrafish, we show that loss of Strip1 causes defects in IPL formation. In strip1 mutants, RGCs undergo dramatic cell death shortly after birth. AC and BP cells subsequently invade the degenerating RGC layer, leading to a disorganized IPL. Mechanistically, zebrafish Strip1 interacts with its STRIPAK partner, Striatin 3 (Strn3), and both show overlapping functions in RGC survival. Furthermore, loss of Strip1 or Strn3 leads to activation of the proapoptotic marker, Jun, within RGCs, and Jun knockdown rescues RGC survival in strip1 mutants. In addition to its function in RGC maintenance, Strip1 is required for RGC dendritic patterning, which likely contributes to proper IPL formation. Taken together, we propose that a series of Strip1-mediated regulatory events coordinates inner retinal circuit formation by maintaining RGCs during development, which ensures proper positioning and neurite patterning of inner retinal neurons.

Absence of Connexin 43 Results in Smaller Retinas and Arrested, Depolarized Retinal Progenitor Cells in Human Retinal Organoids.

The development of the vertebrate retina relies on complex regulatory mechanisms to achieve its characteristic layered morphology containing multiple neuronal cell types. While connexin 43 (CX43) is not expressed by mature retinal neurons, mutations in its gene GJA1 are associated with microphthalmia and low vision in patients. To delineate how lack of CX43 affects retinal development, GJA1 was disrupted in human induced pluripotent stem cells (hiPSCs) (GJA1-/-) using CRISPR/Cas9 editing, and these were subsequently differentiated into retinal organoids. GJA1-/- hiPSCs do not display defects in self-renewal and pluripotency, but the resulting organoids are smaller with a thinner neural retina and decreased abundance of many retinal cell types. CX43-deficient organoids express lower levels of the neural marker PAX6 and the retinal progenitor cell (RPC) markers PAX6, SIX3, and SIX6. Conversely, expression of the early neuroectoderm markers SOX1 and SOX2 remains high in GJA1-/- organoids throughout their development. The lack of CX43 results in an increased population of CHX10-positive RPCs that are smaller, disorganized, do not become polarized, and possess a limited ability to commit to retinal fate specification. Our data indicate that lack of CX43 causes a developmental arrest in RPCs that subsequently leads to pan-retinal defects and stunted ocular growth.

Simple model for encoding natural images by retinal ganglion cells with nonlinear spatial integration.

A central goal in sensory neuroscience is to understand the neuronal signal processing involved in the encoding of natural stimuli. A critical step towards this goal is the development of successful computational encoding models. For ganglion cells in the vertebrate retina, the development of satisfactory models for responses to natural visual scenes is an ongoing challenge. Standard models typically apply linear integration of visual stimuli over space, yet many ganglion cells are known to show nonlinear spatial integration, in particular when stimulated with contrast-reversing gratings. We here study the influence of spatial nonlinearities in the encoding of natural images by ganglion cells, using multielectrode-array recordings from isolated salamander and mouse retinas. We assess how responses to natural images depend on first- and second-order statistics of spatial patterns inside the receptive field. This leads us to a simple extension of current standard ganglion cell models. We show that taking not only the weighted average of light intensity inside the receptive field into account but also its variance over space can partly account for nonlinear integration and substantially improve response predictions of responses to novel images. For salamander ganglion cells, we find that response predictions for cell classes with large receptive fields profit most from including spatial contrast information. Finally, we demonstrate how this model framework can be used to assess the spatial scale of nonlinear integration. Our results underscore that nonlinear spatial stimulus integration translates to stimulation with natural images. Furthermore, the introduced model framework provides a simple, yet powerful extension of standard models and may serve as a benchmark for the development of more detailed models of the nonlinear structure of receptive fields.

Photo-activation of mitochondrial ATP synthesis.

ATP production by mitochondria isolated from Saccharomyces cerevisiae cells was accelerated upon both direct and indirect mitochondrial photo-activation (MPA). The extent of direct MPA was dependent on the wavelength of excitation light. Direct MPA was created by light in cytochrome c spectral absorption bands (440, 520 and 550nm), this light was absorbed producing electronically excited cytochrome c, and the excitation energy of the latter was used in the ATP production chain. The activity of cytochrome c was tested with 600nm light, where cytochrome c does not absorb, and thus ATP production rate remained the same as in darkness. Note that ATP production rates were significantly larger under light at 550, 520 and 440nm. Therefore, photo-activation of cytochrome c was the first step of MPA synthesis of ATP. Indirect MPA of ATP production also proceeded via electronically excited cytochrome c, by energy transfer from electronically excited Co/BN film to cytochrome c located in the inner mitochondrial membrane (IMM). Co/BN excitons were generated by photons absorbed by the Co/BN film, which was not in contact with the mitochondrial sample. Next, these excitons propagated along the Co/BN film to the part of the film that was in contact with the mitochondrial sample. There the exciton energy was transferred to cytochrome c located in the IMM, producing electronically excited cytochrome c. Thus, excited cytochrome c was generated in a way different from that of direct MPA. Next, the energy of excited cytochrome c was used in activated ATP synthesis, with virtually the same effect for 519 and 427nm excitation. Thus, the first step of ATP synthesis in indirect MPA was the exciton energy transfer from Co/BN film to cytochrome c located in the IMM, producing an electronically excited cytochrome c molecule. A phenomenological mechanism of direct and indirect MPA was proposed, and the model parameters were obtained by fitting the model to the experimental data. However, more information is needed before the detailed mechanism of ATP synthesis activation by electronically excited cytochrome c could be understood. The present results support the earlier proposed hypothesis of indirect MPA of ATP production in vertebrate retina in daylight.

Müller glia fused with adult stem cells undergo neural differentiation in human retinal models

SummaryBackgroundVisual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.MethodsWe used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.FindingsWe first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.InterpretationWe show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.FundingThis work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).

Fatty acid-binding proteins and fatty acid synthase influence glial reactivity and promote the formation of Müller glia-derived progenitor cells in the chick retina.

A recent comparative transcriptomic study of Müller glia (MG) in vertebrate retinas revealed that fatty acid binding proteins (FABPs) are among the most highly expressed genes in chick ( Hoang et al., 2020). Here, we investigate how FABPs and fatty acid synthase (FASN) influence glial cells in the chick retina. During development, FABP7 is highly expressed by retinal progenitor cells and maturing MG, whereas FABP5 is upregulated in maturing MG. PMP2 (FABP8) is expressed by oligodendrocytes and FABP5 is expressed by non-astrocytic inner retinal glial cells, and both of these FABPs are upregulated by activated MG. In addition to suppressing the formation of Müller glia-derived progenitor cells (MGPCs), we find that FABP-inhibition suppresses the proliferation of microglia. FABP-inhibition induces distinct changes in single cell transcriptomic profiles, indicating transitions of MG from resting to reactive states and suppressed MGPC formation, with upregulation of gene modules for gliogenesis and decreases in neurogenesis. FASN-inhibition increases the proliferation of microglia and suppresses the formation of MGPCs. We conclude that fatty acid metabolism and cell signaling involving fatty acids are important in regulating the reactivity and dedifferentiation of MG, and the proliferation of microglia and MGPCs.

Reproducibility of the Rod Photoreceptor Response Depends Critically on the Concentration of the Phosphodiesterase Effector Enzyme.

The high sensitivity of night vision requires that rod photoreceptors reliably and reproducibly signal the absorption of single photons, a process that depends on tight regulation of intracellular cGMP concentration through the phototransduction cascade. Here in the mouse (Mus musculus), we studied a single-site D167A mutation of the gene for the α subunit of rod photoreceptor phosphodiesterase (PDEA), made with the aim of removing a noncatalytic binding site for cGMP. This mutation unexpectedly eliminated nearly all PDEA expression and reduced expression of the β subunit (PDEB) to ∼5%-10% of WT. The remaining PDE had nearly normal specific activity; degeneration was slow, with 50%-60% of rods remaining after 6 months. Responses were larger and more sensitive than normal but slower in rise and decay, probably from slower dark turnover of cGMP. Remarkably, responses became much less reproducible than WT, with response variance increasing for amplitude by over 10-fold, and for latency and time-to-peak by >100-fold. We hypothesize that the increase in variance is the result of greater variability in the dark-resting concentration of cGMP, produced by spatial and temporal nonuniformity in spontaneous PDE activity. This variability decreased as stimuli were made brighter, presumably because of greater spatial uniformity of phototransduction and the approach to saturation. We conclude that the constancy of the rod response depends critically on PDE expression to maintain adequate spontaneous PDE activity, so that the concentration of second messenger is relatively uniform throughout the outer segment.SIGNIFICANCE STATEMENT Rod photoreceptors in the vertebrate retina reliably signal the absorption of single photons of light by generating responses that are remarkably reproducible in amplitude and waveform. We show that this reproducibility depends critically on the concentration of the effector enzyme phosphodiesterase (PDE), which metabolizes the second messenger cGMP and generates rod light responses. In rods with the D167A mutation of the α subunit of PDE, only 5%-10% of PDE is expressed. Single-photon responses then become much more variable than in WT rods. We think this variability is caused by spatial and temporal inhomogeneity in the concentration of cGMP in darkness, so that photons absorbed in different parts of the cell produce responses of greatly varying amplitude and waveform.