Plasticity of cone photoreceptors in adult zebrafish revealed by thyroid hormone exposure.

Vertebrate color vision is predominantly mediated by the presence of multiple cone photoreceptor subtypes that are each maximally sensitive to different wavelengths of light. Thyroid hormone (TH) has been shown to be essential in the spatiotemporal patterning of cone subtypes in many species, including cone subtypes that express opsins that are encoded by tandemly replicated genes. TH has been shown to differentially regulate the tandemly replicated lws opsin genes in zebrafish, and exogenous treatments alter the expression levels of these genes in larvae and juveniles. In this study, we sought to determine whether gene expression in cone photoreceptors remains plastic to TH treatment in adults. We used a transgenic lws reporter line, multiplexed fluorescence hybridization chain reaction in situ hybridization, and qPCR to examine the extent to which cone gene expression can be altered by TH in adults. Our studies revealed that opsin gene expression, and the expression of other photoreceptor genes, remains plastic to TH treatment in adult zebrafish. In addition to retinal plasticity, exogenous TH treatment alters skin pigmentation patterns in adult zebrafish after 5 days. Taken together, our results show a remarkable level of TH-sensitive plasticity in the adult zebrafish.

Long wavelength-sensing cones of zebrafish retina exhibit multiple layers of transcriptional heterogeneity.

Introduction: Understanding how photoreceptor genes are regulated is important for investigating retinal development and disease. While much is known about gene regulation in cones, the mechanism by which tandemly-replicated opsins, such as human long wavelength-sensitive and middle wavelength-sensitive opsins, are differentially regulated remains elusive. In this study, we aimed to further our understanding of transcriptional heterogeneity in cones that express tandemly-replicated opsins and the regulation of such differential expression using zebrafish, which express the tandemly-replicated opsins lws1 and lws2. Methods: We performed bulk and single cell RNA-Seq of LWS1 and LWS2 cones, evaluated expression patterns of selected genes of interest using multiplex fluorescence in situ hybridization, and used exogenous thyroid hormone (TH) treatments to test selected genes for potential control by thyroid hormone: a potent, endogenous regulator of lws1 and lws2 expression. Results: Our studies indicate that additional transcriptional differences beyond opsin expression exist between LWS1 and LWS2 cones. Bulk RNA-Seq results showed 95 transcripts enriched in LWS1 cones and 186 transcripts enriched in LWS2 cones (FC > 2, FDR < 0.05). In situ hybridization results also reveal underlying heterogeneity within the lws1- and lws2-expressing populations. This heterogeneity is evident in cones of mature zebrafish, and further heterogeneity is revealed in transcriptional responses to TH treatments. Discussion: We found some evidence of coordinate regulation of lws opsins and other genes by exogenous TH in LWS1 vs. LWS2 cones, as well as evidence of gene regulation not mediated by TH. The transcriptional differences between LWS1 and LWS2 cones are likely controlled by multiple signals, including TH.

Generating Widespread and Scalable Retinal Lesions in Adult Zebrafish by Intraocular Injection of Ouabain.

Zebrafish regenerate functional retinal neurons after injury. Regeneration takes place following photic, chemical, mechanical, surgical, or cryogenic lesions, as well as after lesions that selectively target specific neuronal cell populations. An advantage of chemical retinal lesion for studying the process of regeneration is that the lesion is topographically widespread. This results in the loss of visual function as well as a regenerative response that engages nearly all stem cells (Müller glia). Such lesions can therefore be used to further our understanding of the process and mechanisms underlying re-establishment of neuronal wiring patterns, retinal function, and visually mediated behaviors. Widespread chemical lesions also permit the quantitative analysis of gene expression throughout the retina during the period of initial damage and over the duration of regeneration, as well as the study of growth and targeting of axons of regenerated retinal ganglion cells. The neurotoxic Na+/K+ ATPase inhibitor ouabain specifically offers a further advantage over other types of chemical lesions in that it is scalable; the extent of damage can be targeted to include only inner retinal neurons, or all retinal neurons, simply by adjusting the intraocular concentration of ouabain that is used. Here we describe the procedure through which these "selective" vs. "extensive" retinal lesions can be generated.

Assessing Rewiring of the Retinal Circuitry by Electroretinogram (ERG) After Inner Retinal Lesion in Adult Zebrafish.

Adult zebrafish respond to retinal injury with a regenerative response that replaces damaged neurons with Müller glia-derived regenerated neurons. The regenerated neurons are functional, appear to make appropriate synaptic connections, and support visually mediated reflexes and more complex behaviors. Curiously, the electrophysiology of damaged, regenerating, and regenerated zebrafish retina has only recently been examined. In our previous work, we demonstrated that electroretinogram (ERG) recordings of damaged zebrafish retina correlate with the extent of the inflicted damage and that the regenerated retina at 80 days post-injury exhibited ERG waveforms consistent with functional visual processing. In this paper we describe the procedure for obtaining and analyzing ERG recordings from adult zebrafish previously subjected to widespread lesions that destroy inner retinal neurons and engage a regenerative response that restores retinal function, in particular the synaptic connections between photoreceptor axon terminals and the dendritic trees of retinal bipolar neurons.

Dynamic functional and structural remodeling during retinal regeneration in zebrafish.

Introduction: Zebrafish regenerate their retinas following damage, resulting in restoration of visual function. Here we evaluate recovery of retinal function through qualitative and quantitative analysis of the electroretinogram (ERG) over time following retinal damage, in correlation to histological features of regenerated retinal tissue. Methods: Retinas of adult zebrafish were lesioned by intravitreal injection of 10 µM (extensive lesion; destroys all neurons) or 2 µM (selective lesion; spares photoreceptors) ouabain. Unlesioned contralateral retinas served as controls. Function of retinal circuitry was analyzed at selected timepoints using ERG recordings from live zebrafish, and whole eyes were processed for histological analyses immediately thereafter. Results: Qualitative and quantitative assessment of waveforms during retinal regeneration revealed dynamic changes that were heterogeneous on an individual level within each sampling time, but still followed common waveform recovery patterns on a per-fish and population-level basis. Early in the regeneration period (13-30 days post injury; DPI), for both lesion types, b-waves were essentially not detected, and unmasked increased apparent amplitudes, implicit times, and half-widths of a-waves (vs. controls). In control recordings, d-waves were not obviously detected, but apparent d-waves (OFF-bipolar responses) from regenerating retinas of several fish became prominent by 30DPI and dominated the post-photoreceptor response (PPR). Beyond 45DPI, b-waves became detectable, and the ratio of apparent d- to b-wave contributions progressively shifted with most, but not all, fish displaying a b-wave dominated PPR. At the latest timepoints (extensive, 90DPI; selective, 80DPI), recordings with measurable b-waves approached a normal waveform (implicit times and half-widths), but amplitudes were not restored to control levels. Histological analyses of the retinas from which ERGs were recorded showed that as regeneration progressed, PKCa + ON-bipolar terminals and parvalbumin + amacrine cell processes became more stereotypically positioned within the deep sublaminae of the INL over recovery time after each lesion type, consistent with the shift in PPR seen in the ERG recordings. Discussion: Taken together, these data suggest that photoreceptor-OFF-bipolar component/connectivity may functionally recover and mature earlier during regeneration compared to the photoreceptor-ON-bipolar component, though the timeframe in which such recovery happens is heterogeneous on a per-fish basis. Collectively our studies suggest gradual restoration of ON-bipolar functional circuitry during retinal regeneration.

Eye flukes (Diplostomum spp) damage retinal tissue and may cause a regenerative response in wild threespine stickleback fish.

Fish rely upon vision as a dominant sensory system for foraging, predator avoidance, and mate selection. Damage to the visual system, in particular to the neural retina of the eye, has been demonstrated to result in a regenerative response in captive fish that serve as model organisms (e.g. zebrafish), and this response restores some visual function. The purpose of the present study is to determine whether damage to the visual system that occurs in wild populations of fish also results in a regenerative response, offering a potentially ecologically relevant model of retinal regeneration. Adult threespine stickleback were collected from several water bodies of Iceland, and cryosectioned eye tissues were processed for hematoxylin and eosin staining or for indirect immunofluorescence using cell-specific markers. In many of the samples, eye flukes (metacercariae of Diplostomum spp) were present, frequently between the neural retina and retinal pigmented epithelium (RPE). Damage to the retina and to the RPE was evident in eyes containing flukes, and RPE fragments were observed within fluke bodies, suggesting they had consumed this eye tissue. Expression of a cell proliferation marker was also observed in both retina and RPE, consistent with a proliferative response to the damage. Interestingly, some regions of infected retina displayed "laminar fusions," in which neuronal cell bodies were misplaced within the major synaptic layer of the retina. These laminar fusions are also frequently found in regenerated zebrafish retina following non-parasitic (experimental) forms of retinal damage. The stickleback retina may therefore respond to fluke-mediated damage by engaging in retinal regeneration.

An EvoDevo Study of Salmonid Visual Opsin Dynamics and Photopigment Spectral Sensitivity.

Salmonids are ideal models as many species follow a distinct developmental program from demersal eggs and a large yolk sac to hatching at an advanced developmental stage. Further, these economically important teleosts inhabit both marine- and freshwaters and experience diverse light environments during their life histories. At a genome level, salmonids have undergone a salmonid-specific fourth whole genome duplication event (Ss4R) compared to other teleosts that are already more genetically diverse compared to many non-teleost vertebrates. Thus, salmonids display phenotypically plastic visual systems that appear to be closely related to their anadromous migration patterns. This is most likely due to a complex interplay between their larger, more gene-rich genomes and broad spectrally enriched habitats; however, the molecular basis and functional consequences for such diversity is not fully understood. This study used advances in genome sequencing to identify the repertoire and genome organization of visual opsin genes (those primarily expressed in retinal photoreceptors) from six different salmonids [Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Chinook salmon (Oncorhynchus tshawytcha), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), and sockeye salmon (Oncorhynchus nerka)] compared to the northern pike (Esox lucius), a closely related non-salmonid species. Results identified multiple orthologues for all five visual opsin classes, except for presence of a single short-wavelength-sensitive-2 opsin gene. Several visual opsin genes were not retained after the Ss4R duplication event, which is consistent with the concept of salmonid rediploidization. Developmentally, transcriptomic analyzes of Atlantic salmon revealed differential expression within each opsin class, with two of the long-wavelength-sensitive opsins not being expressed before first feeding. Also, early opsin expression in the retina was located centrally, expanding dorsally and ventrally as eye development progressed, with rod opsin being the dominant visual opsin post-hatching. Modeling by spectral tuning analysis and atomistic molecular simulation, predicted the greatest variation in the spectral peak of absorbance to be within the Rh2 class, with a ∼40 nm difference in λ max values between the four medium-wavelength-sensitive photopigments. Overall, it appears that opsin duplication and expression, and their respective spectral tuning profiles, evolved to maximize specialist color vision throughout an anadromous lifecycle, with some visual opsin genes being lost to tailor marine-based vision.

Evidence of regional specializations in regenerated zebrafish retina.

Adult zebrafish are capable of functional retinal regeneration following damage. A goal of vision science is to stimulate or permit a similar process in mammals to treat human retinal disease and trauma. Ideally such a process would reconstitute the stereotyped, two-dimensional topographic patterns and regional specializations of specific cell types, functionally important for representation of the visual field. An example in humans is the cone-rich fovea, essential for high-acuity color vision. Stereotyped, global topographic patterns of specific retinal cell types are also found in zebrafish, particularly for cone types expressing the tandemly-replicated lws (long wavelength-sensitive) and rh2 (middle wavelength-sensitive) opsins. Here we examine whether regionally specialized patterns of LWS1 and LWS2 cones are restored in regenerated retinas in zebrafish. Adult transgenic zebrafish carrying fluorescent reporters for lws1 and lws2 were subjected to retinal lesions that destroy all neurons but spare glia, via intraocular injection of the neurotoxin ouabain. Regenerated and contralateral control retinas were mounted whole or sectioned, and imaged. Overall spatial patterns of lws1 vs. lws2 opsin-expressing cones in regenerated retinas were remarkably similar to those of control retinas, with LWS1 cones in ventral/peripheral regions, and LWS2 cones in dorsal/central regions. However, LWS2 cones occupied a smaller fraction of regenerated retina, and several cones co-expressed the lws1 and lws2 reporters in regenerated retinas. Local patterns of regenerated LWS1 cones showed modest reductions in regularity. These results suggest that some of the regional patterning information, or the source of such signals, for LWS cone subtypes may be retained by undamaged cell types (Müller glia or RPE) and re-deployed during regeneration.

Selective Requirements for Vascular Endothelial Cells and Circulating Factors in the Regulation of Retinal Neurogenesis.

Development of the vertebrate eye requires signaling interactions between neural and non-neural tissues. Interactions between components of the vascular system and the developing neural retina have been difficult to decipher, however, due to the challenges of untangling these interactions from the roles of the vasculature in gas exchange. Here we use the embryonic zebrafish, which is not yet reliant upon hemoglobin-mediated oxygen transport, together with genetic strategies for (1) temporally-selective depletion of vascular endothelial cells, (2) elimination of blood flow through the circulation, and (3) elimination of cells of the erythroid lineage, including erythrocytes. The retinal phenotypes in these genetic systems were not identical, with endothelial cell-depleted retinas displaying laminar disorganization, cell death, reduced proliferation, and reduced cell differentiation. In contrast, the lack of blood flow resulted in a milder retinal phenotype showing reduced proliferation and reduced cell differentiation, indicating that an endothelial cell-derived factor(s) is/are required for laminar organization and cell survival. The lack of erythrocytes did not result in an obvious retinal phenotype, confirming that defects in retinal development that result from vascular manipulations are not due to poor gas exchange. These findings underscore the importance of the cardiovascular system supporting and controlling retinal development in ways other than supplying oxygen. In addition, these findings identify a key developmental window for these interactions and point to distinct functions for vascular endothelial cells vs. circulating factors.

Short-wavelength-sensitive 2 (Sws2) visual photopigment models combined with atomistic molecular simulations to predict spectral peaks of absorbance.

For many species, vision is one of the most important sensory modalities for mediating essential tasks that include navigation, predation and foraging, predator avoidance, and numerous social behaviors. The vertebrate visual process begins when photons of the light interact with rod and cone photoreceptors that are present in the neural retina. Vertebrate visual photopigments are housed within these photoreceptor cells and are sensitive to a wide range of wavelengths that peak within the light spectrum, the latter of which is a function of the type of chromophore used and how it interacts with specific amino acid residues found within the opsin protein sequence. Minor differences in the amino acid sequences of the opsins are known to lead to large differences in the spectral peak of absorbance (i.e. the λmax value). In our prior studies, we developed a new approach that combined homology modeling and molecular dynamics simulations to gather structural information associated with chromophore conformation, then used it to generate statistical models for the accurate prediction of λmax values for photopigments derived from Rh1 and Rh2 amino acid sequences. In the present study, we test our novel approach to predict the λmax of phylogenetically distant Sws2 cone opsins. To build a model that can predict the λmax using our approach presented in our prior studies, we selected a spectrally-diverse set of 11 teleost Sws2 photopigments for which both amino acid sequence information and experimentally measured λmax values are known. The final first-order regression model, consisting of three terms associated with chromophore conformation, was sufficient to predict the λmax of Sws2 photopigments with high accuracy. This study further highlights the breadth of our approach in reliably predicting λmax values of Sws2 cone photopigments, evolutionary-more distant from template bovine RH1, and provided mechanistic insights into the role of known spectral tuning sites.

Endocrine regulation of multichromatic color vision.

Vertebrate color vision requires spectrally selective opsin-based pigments, expressed in distinct cone photoreceptor populations. In primates and in fish, spectrally divergent opsin genes may reside in head-to-tail tandem arrays. Mechanisms underlying differential expression from such arrays have not been fully elucidated. Regulation of human red (LWS) vs. green (MWS) opsins is considered a stochastic event, whereby upstream enhancers associate randomly with promoters of the proximal or distal gene, and one of these associations becomes permanent. We demonstrate that, distinct from this stochastic model, the endocrine signal thyroid hormone (TH) regulates differential expression of the orthologous zebrafish lws1/lws2 array, and of the tandemly quadruplicated rh2-1/rh2-2/rh2-3/rh2-4 array. TH treatment caused dramatic, dose-dependent increases in abundance of lws1, the proximal member of the lws array, and reduced lws2 Fluorescent lws reporters permitted direct visualization of individual cones switching expression from lws2 to lws1 Athyroidism increased lws2 and reduced lws1, except within a small ventral domain of lws1 that was likely sustained by retinoic acid signaling. Changes in lws abundance and distribution in athyroid zebrafish were rescued by TH, demonstrating plasticity of cone phenotype in response to this signal. TH manipulations also regulated the rh2 array, with athyroidism reducing abundance of distal members. Interestingly, the opsins encoded by the proximal lws gene and distal rh2 genes are sensitive to longer wavelengths than other members of their respective arrays; therefore, endogenous TH acts upon each opsin array to shift overall spectral sensitivity toward longer wavelengths, underlying coordinated changes in visual system function during development and growth.

Rewiring the Regenerated Zebrafish Retina: Reemergence of Bipolar Neurons and Cone-Bipolar Circuitry Following an Inner Retinal Lesion.

We previously reported strikingly normal morphologies and functional connectivities of regenerated retinal bipolar neurons (BPs) in zebrafish retinas sampled 60 days after a ouabain-mediated lesion of inner retinal neurons (60 DPI) (McGinn et al., 2018). Here we report early steps in the birth of BPs and formation of their dendritic trees and axonal arbors during regeneration. Adult zebrafish were subjected to ouabain-mediated lesion that destroys inner retinal neurons but spares photoreceptors and Müller glia, and were sampled at 13, 17, and 21 DPI, a timeframe over which plexiform layers reemerge. We show that this timeframe corresponds to reemergence of two populations of BPs (PKCα+ and nyx::mYFP+). Sequential BrdU, EdU incorporation reveals that similar fractions of PKCα+ BPs and HuC/D+ amacrine/ganglion cells are regenerated concurrently, suggesting that the sequence of neuronal production during retinal regeneration does not strictly match that observed during embryonic development. Further, accumulation of regenerated BPs appears protracted, at least through 21 DPI. The existence of isolated, nyx::mYFP+ BPs allowed examination of cytological detail through confocal microscopy, image tracing, morphometric analyses, identification of cone synaptic contacts, and rendering/visualization. Apically-projecting neurites (=dendrites) of regenerated BPs sampled at 13, 17, and 21 DPI are either truncated, or display smaller dendritic trees when compared to controls. In cases where BP dendrites reach the outer plexiform layer (OPL), numbers of dendritic tips are similar to those of controls at all sampling times. Further, by 13-17 DPI, BPs with dendritic tips reaching the outer nuclear layer (ONL) show patterns of photoreceptor connections that are statistically indistinguishable from controls, while those sampled at 21 DPI slightly favor contacts with double cone synaptic terminals over those of blue-sensitive cones. These findings suggest that once regenerated BP dendrites reach the OPL, normal photoreceptor connectomes are established, albeit with some plasticity. Through 17 DPI, some basally-projecting neurites (=axons) of regenerated nyx::mYFP+ BPs traverse long distances, branch into inappropriate layers, or appear to abruptly terminate. These findings suggest that, after a tissue-disrupting lesion, regeneration of inner retinal neurons is a dynamic process that includes ongoing genesis of new neurons and changes in BP morphology.

Vision using multiple distinct rod opsins in deep-sea fishes.

Vertebrate vision is accomplished through light-sensitive photopigments consisting of an opsin protein bound to a chromophore. In dim light, vertebrates generally rely on a single rod opsin [rhodopsin 1 (RH1)] for obtaining visual information. By inspecting 101 fish genomes, we found that three deep-sea teleost lineages have independently expanded their RH1 gene repertoires. Among these, the silver spinyfin (Diretmus argenteus) stands out as having the highest number of visual opsins in vertebrates (two cone opsins and 38 rod opsins). Spinyfins express up to 14 RH1s (including the most blueshifted rod photopigments known), which cover the range of the residual daylight as well as the bioluminescence spectrum present in the deep sea. Our findings present molecular and functional evidence for the recurrent evolution of multiple rod opsin-based vision in vertebrates.

Regeneration associated transcriptional signature of retinal microglia and macrophages.

Zebrafish have the remarkable capacity to regenerate retinal neurons following a variety of damage paradigms. Following initial tissue insult and a period of cell death, a proliferative phase ensues that generates neuronal progenitors, which ultimately regenerate damaged neurons. Recent work has revealed that Müller glia are the source of regenerated neurons in zebrafish. However, the roles of another important class of glia present in the retina, microglia, during this regenerative phase remain elusive. Here, we examine retinal tissue and perform QuantSeq. 3'mRNA sequencing/transcriptome analysis to reveal localization and putative functions, respectively, of mpeg1 expressing cells (microglia/macrophages) during Müller glia-mediated regeneration, corresponding to a time of progenitor proliferation and production of new neurons. Our results indicate that in this regenerative state, mpeg1-expressing cells are located in regions containing regenerative Müller glia and are likely engaged in active vesicle trafficking. Further, mpeg1+ cells congregate at and around the optic nerve head. Our transcriptome analysis reveals several novel genes not previously described in microglia. This dataset represents the first report, to our knowledge, to use RNA sequencing to probe the microglial transcriptome in such context, and therefore provides a resource towards understanding microglia/macrophage function during successful retinal (and central nervous tissue) regeneration.

Isolation of photoreceptors from mature, developing, and regenerated zebrafish retinas, and of microglia/macrophages from regenerating zebrafish retinas.

This paper describes experimental procedures for the dissociation of retinal cells of the zebrafish (Danio rerio) for subsequent fluorescence-activated cell sorting (FACS) and gene expression studies. Methods for dissociation of zebrafish retinas followed by FACS and RNA isolation were optimized. This methodology was applied to isolate pure sorted samples of rods, long wavelength-sensitive (LWS) cones, medium wavelength-sensitive (MWS; RH2-2) cones, short wavelength-sensitive (SWS2) cones, and UV-sensitive (SWS1) cones from retinas obtained at selective life-history stages of the zebrafish, and for some of these photoreceptors, following retinal regeneration. We also successfully separated lws1-expressing and lws2-expressing LWS cones from fish of a transgenic line in which lws1 is reported with green fluorescence protein (GFP) and lws2 is reported with red fluorescence protein (RFP). Microglia/macrophages were successfully sorted from regenerating retinas (7 days after a cytotoxic lesion) of a transgenic line in which these immune cells express GFP. Electropherograms verified downstream isolation of high-quality RNA from sorted samples. Examples of post-sorting analysis, as well as results of qRT-PCR studies, validated the purity of sorted populations. For example, qRT-PCR samples derived from isolated Rh2-2 cones contained detectable rh2-2 (opn1mw2) opsin transcripts, but lws opsin transcripts (lws1/opn1lw1, lws2/opn1lw2) were not detected, suggesting that the procedure likely separated double cone pairs. Through this method, pure, sorted cell samples can provide RNA that is reliable for downstream gene expression analyses, such as qRT-PCR and RNA-seq, which may reveal molecular signatures of photoreceptors and microglia for comparative transcriptomics studies.

Expression patterns of dscam and sdk gene paralogs in developing zebrafish retina.

Purpose: The differential adhesion hypothesis states that a cell adhesion code provides cues that direct the specificity of nervous system development. The Down syndrome cell adhesion molecule (DSCAM) and sidekick (SDK) proteins belong to the immunoglobulin superfamily of cell adhesion molecules (CAMs) and provide both attractive and repulsive cues that help to organize the nervous system during development, according to the differential adhesion hypothesis. The zebrafish genome is enriched in dscam and sdk genes, making the zebrafish an excellent model system to further test this hypothesis. The goal of this study is to describe the phylogenetic relationships of the paralogous CAM genes and their spatial expression and co-expression patterns in the embryonic zebrafish retina. Methods: Exon-intron structures, karyotypic locations, genomic context, and amino acid sequences of the zebrafish CAM genes (dscama, dscamb, dscaml1, sdk1a, sdk1b, sdk2a, and sdk2b) were obtained from the Ensembl genome database. The Prosite and SMART programs were used to determine the number and identity of protein domains for each CAM gene. The randomized axelerated maximum likelihood (RaxML) program was used to perform a phylogenetic analysis of the zebrafish CAM genes and orthologs in other vertebrates. A synteny analysis of regions surrounding zebrafish CAM paralogs was performed. Digoxigenin (dig)-labeled cRNA probes for each CAM gene were generated to perform in situ hybridization of retinal cryosections from zebrafish embryos and larvae. Dual in situ hybridization of retinal cryosections from zebrafish larvae was performed with dig- and fluorescein-labeled cRNA probes. Results: We found the studied zebrafish CAM genes encode similar protein domain structures as their corresponding orthologs in mammals and possess similar intron-exon organizations. CAM paralogs were located on different chromosomes. Phylogenetic and synteny analyses provided support for zebrafish dscam and sdk2 paralogs having originated during the teleost genome duplication. We found that dscama and dscamb are co-expressed in the ganglion cell layer (GCL) and the basal portion of the inner nuclear layer (INL), with weak expression in the photoreceptor-containing outer nuclear layer (ONL). Of the dscam genes, only dscamb was strongly expressed in ONL. Sdk1a and sdk1b were co-expressed in the GCL and the basal portion of the INL. Sdk2a and sdk2b also showed co-expression in the GCL and basal portion of the INL. All Sdk genes were expressed in the ciliary marginal zone (CMZ). Dual in situ hybridizations revealed alternating patterns of co-expression and exclusive expression for the dscam and sdk1 paralogs in cells of the GCL and the INL. The same alternating pattern was observed between dscam and sdk2 paralogs and between sdk1 and sdk2 paralogs. The expression of dscaml1 was observed in the INL and the GCL, with some cells in the basal portion of the INL showing co-expression of dscaml1 and dscama. Conclusions: These findings suggest that zebrafish dscam and sdk2 paralogs were likely the result of the teleost whole genome duplication and that all CAM duplicates show some differential expression patterns. We also demonstrate that the comparative expression patterns of CAM genes in the zebrafish are distinct from the exclusive expression patterns observed in chick retina, in which retinal ganglion cells express one of the four chick Dscam or Sdk genes only. The patterns in zebrafish are more similar to those of mice, in which co-expression of Dscam and Sdk genes is observed. These findings provide the groundwork for future functional analysis of the roles of the CAM paralogs in zebrafish.

Dynamic changes in microglial and macrophage characteristics during degeneration and regeneration of the zebrafish retina.

BACKGROUND: In contrast to mammals, zebrafish have the capacity to regenerate retinal neurons following a variety of injuries. Two types of glial cells, Müller glia (MG) and microglia, are known to exist in the zebrafish retina. Recent work has shown that MG give rise to regenerated retinal neurons, but the role of resident microglia, and the innate immune system more generally, during retinal regeneration is not well defined. Specifically, characteristics of the immune system and microglia following substantial neuron death and a successful regenerative response have not been documented. METHODS: The neurotoxin ouabain was used to induce a substantial retinal lesion of the inner retina in zebrafish. This lesion results in a regenerative response that largely restores retinal architecture, neuronal morphologies, and connectivities, as well as recovery of visual function. We analyzed cryosections from damaged eyes following immunofluorescence and H&E staining to characterize the initial immune response to the lesion. Whole retinas were analyzed by confocal microscopy to characterize microglia morphology and distribution. Statistical analysis was performed using a two-tailed Student's t test comparing damaged to control samples. RESULTS: We find evidence of early leukocyte infiltration to the retina in response to ouabain injection followed by a period of immune cell proliferation that likely includes both resident microglia and substantial numbers of proliferating, extra-retinally derived macrophages, leading to rapid accumulation upon retinal damage. Following immune cell proliferation, Müller glia re-enter the cell cycle. In retinas that have regenerated the layers lost to the initial injury (histologically regenerated), microglia retain morphological features of activation, suggesting ongoing functions that are likely essential to restoration of retinal function. CONCLUSIONS: Collectively, these results indicate that microglia and the immune system are dynamic during a successful regenerative response in the retina. This study provides an important framework to probe inflammation in the initiation of, and functional roles of microglia during retinal regeneration.

Transcripts within rod photoreceptors of the Zebrafish retina.

BACKGROUND: The purpose of this study was to identify transcripts of retinal rod photoreceptors of the zebrafish. The zebrafish is an important animal model for vision science due to rapid and tractable development, persistent neurogenesis of rods throughout the lifespan, and capacity for functional retinal regeneration. RESULTS: Zebrafish rods, and non-rod retinal cells of the xops:eGFP transgenic line, were separated by cell dissociation and fluorescence-activated cell sorting (FACS), followed by RNA-seq. At a false discovery rate of < 0.01, 597 transcripts were upregulated ("enriched") in rods vs. other retinal cells, and 1032 were downregulated ("depleted"). Thirteen thousand three hundred twenty four total transcripts were detected in rods, including many not previously known to be expressed by rods. Forty five transcripts were validated by qPCR in FACS-sorted rods vs. other retinal cells. Transcripts enriched in rods from adult retinas were also enriched in rods from larval and juvenile retinas, and were also enriched in rods sorted from retinas subjected to a neurotoxic lesion and allowed to regenerate. Many transcripts enriched in rods were upregulated in retinas of wildtype retinas vs. those of a zebrafish model for rod degeneration. CONCLUSIONS: We report the generation and validation of an RNA-seq dataset describing the rod transcriptome of the zebrafish, which is now available as a resource for further studies of rod photoreceptor biology and comparative transcriptomics.

Predicting peak spectral sensitivities of vertebrate cone visual pigments using atomistic molecular simulations.

Vision is the dominant sensory modality in many organisms for foraging, predator avoidance, and social behaviors including mate selection. Vertebrate visual perception is initiated when light strikes rod and cone photoreceptors within the neural retina of the eye. Sensitivity to individual colors, i.e., peak spectral sensitivities (λmax) of visual pigments, are a function of the type of chromophore and the amino acid sequence of the associated opsin protein in the photoreceptors. Large differences in peak spectral sensitivities can result from minor differences in amino acid sequence of cone opsins. To determine how minor sequence differences could result in large spectral shifts we selected a spectrally-diverse group of 14 teleost Rh2 cone opsins for which sequences and λmax are experimentally known. Classical molecular dynamics simulations were carried out after embedding chromophore-associated homology structures within explicit bilayers and water. These simulations revealed structural features of visual pigments, particularly within the chromophore, that contributed to diverged spectral sensitivities. Statistical tests performed on all the observed structural parameters associated with the chromophore revealed that a two-term, first-order regression model was sufficient to accurately predict λmax over a range of 452-528 nm. The approach was accurate, efficient and simple in that site-by-site molecular modifications or complex quantum mechanics models were not required to predict λmax. These studies identify structural features associated with the chromophore that may explain diverged spectral sensitivities, and provide a platform for future, functionally predictive opsin modeling.

Restoration of Dendritic Complexity, Functional Connectivity, and Diversity of Regenerated Retinal Bipolar Neurons in Adult Zebrafish.

Adult zebrafish (Danio rerio) are capable of regenerating retinal neurons that have been lost due to mechanical, chemical, or light damage. In the case of chemical damage, there is evidence that visually mediated behaviors are restored after regeneration, consistent with recovery of retinal function. However, the extent to which regenerated retinal neurons attain appropriate morphologies and circuitry after such tissue-disrupting lesions has not been investigated. Adult zebrafish of both sexes were subjected to intravitreal injections of ouabain, which destroys the inner retina. After retinal regeneration, cell-selective markers, confocal microscopy, morphometrics, and electrophysiology were used to examine dendritic and axonal morphologies, connectivities, and the diversities of each, as well as retinal function, for a subpopulation of regenerated bipolar neurons (BPs). Although regenerated BPs were reduced in numbers, BP dendritic spreads, dendritic tree morphologies, and cone-bipolar connectivity patterns were restored in regenerated retinas, suggesting that regenerated BPs recover accurate input pathways from surviving cone photoreceptors. Morphological measurements of bipolar axons found that numbers and types of stratifications were also restored; however, the thickness of the inner plexiform layer and one measure of axon branching were slightly reduced after regeneration, suggesting some minor differences in the recovery of output pathways to downstream partners. Furthermore, ERG traces from regenerated retinas displayed waveforms matching those of controls, but with reduced b-wave amplitudes. These results support the hypothesis that regenerated neurons of the adult zebrafish retina are capable of restoring complex morphologies and circuitry, suggesting that complex visual functions may also be restored.SIGNIFICANCE STATEMENT Adult zebrafish generate new retinal neurons after a tissue-disrupting lesion. Existing research does not address whether regenerated neurons of adults successfully reconnect with surrounding neurons and establish complex morphologies and functions. We report that, after a chemical lesion that ablates inner retinal neurons, regenerated retinal bipolar neurons (BPs), although reduced in numbers, reconnected to undamaged cone photoreceptors with correct wiring patterns. Regenerated BPs had complex morphologies similar to those within undamaged retina and a physiological measure of photoreceptor-BP connectivity, the ERG, was restored to a normal waveform. This new understanding of neural connectivity, morphology, and physiology suggests that complex functional processing is possible within regenerated adult retina and offers a system for the future study of synaptogenesis during adult retinal regeneration.