Detection of Zebrafish Retinal Proteins by Infrared Western Blotting

The zebrafish retina is a canonical vertebrate retina. Since the past few years, with the continually growing genetic toolbox and imaging techniques, zebrafish plays a crucial role in retinal research. This protocol describes a method to quantitatively evaluate the expression of Arrestin3a (Arr3a) and G-protein receptor kinase7a (Grk7a) in the adult zebrafish retina at protein levels by infrared fluorescence western blot. Our protocol can be easily adapted to measure protein levels in additional zebrafish tissues

Evolution of visual guanylyl cyclases and their activating proteins with respect to clade and species-specific visual system adaptation

Membrane guanylyl cyclase receptors are important regulators of local cGMP production, critically influencing cell growth and differentiation as well as ion transport, blood pressure and calcium feedback of vertebrate phototransduction. Currently, seven different subtypes of membrane guanylyl cyclase receptors have been characterized. These receptors have tissue specific expression and are activated either by small extracellular ligands, changing CO$_{2}$ concentrations or, in the case of visual guanylyl cyclases, intracellularly interacting Ca$^{2+}$-dependent activating proteins. In this report, we focus on the visual guanylyl cyclase receptors (GCs) GC-E (gucy2d/e) and GC-F (gucy2f) and their activating proteins (GCAP1/2/3; guca1a/b/c). While gucy2d/e has been detected in all analyzed vertebrates, GC-F receptors are missing in several clades (reptiles, birds, and marsupials) and/or individual species. Interestingly, the absence of GC-F in highly visual sauropsida species with up to 4 different cone-opsins is compensated by an increased number of guanylyl cyclase activating proteins, whereas in nocturnal or visually impaired species with reduced spectral sensitivity it is consolidated by the parallel inactivation of these activators. In mammals, the presence of GC-E and GC-F is accompanied by the expression of one to three GCAPs, whereas in lizards and birds, up to five different GCAPs are regulating the activity of the single GC-E visual membrane receptor. In several nearly blind species, a single GC-E enzyme is often accompanied by a single variant of GCAP, suggesting that one cyclase and one activating protein are both sufficient and required for conferring the basic detection of light

Biochemistry and physiology of zebrafish photoreceptors

All vertebrates share a canonical retina with light-sensitive photoreceptors in the outer retina. These photoreceptors are of two kinds: rods and cones, adapted to low and bright light conditions, respectively. They both show a peculiar morphology, with long outer segments, comprised of ordered stacks of disc-shaped membranes. These discs host numerous proteins, many of which contribute to the visual transduction cascade. This pathway converts the light stimulus into a biological signal, ultimately modulating synaptic transmission. Recently, the zebrafish (Danio rerio) has gained popularity for studying the function of vertebrate photoreceptors. In this review, we introduce this model system and its contribution to our understanding of photoreception with a focus on the cone visual transduction cascade

Selective Gene Loss of Visual and Olfactory Guanylyl Cyclase Genes Following the Two Rounds of Vertebrate-Specific Whole-Genome Duplications

Photoreceptors convey visual information and come in two flavors; dim-light and bright-light dedicated rod and cones. Both cell types feature highly specialized phototransduction cascades that convert photonic energy into intracellular signals. Although a substantial amount of phototransduction gene ohnologs are expressed either in rods or cones, visual guanylyl cyclases (GCs) involved in the calcium (Ca2+) dependent feedback regulation of phototransduction are neither rod nor cone specific. The co-existence of visual GCs in both photoreceptor types suggests that specialization of these ohnologs occurred despite their overlapping expression. Here, we analyze gene retention and inactivation patterns of vertebrate visual and closely related olfactory GCs following two rounds (2R) of vertebrate-specific whole-genome duplication events (2R WGD). Although eutherians generally use two visual and one olfactory GC, independent inactivation occurred in some lineages. Sauropsids (birds, lizards, snakes, turtles, and crocodiles) generally have only one visual GC (GC-E). Additionally, turtles (testodes) also lost the olfactory GC (GC-D). Pseudogenization in mammals occurred in specific species/families likely according to functional needs (i.e., many species with reduced vision only have GC-E). Likewise, some species not relying on scent marks lack GC-D, the olfactory GC enzyme. Interestingly, in the case of fish, no species can be found with fewer than three (two visual and one olfactory) genes and the teleost-specific 3R WGD can increase this number to up to five. This suggests that vision in fish now requires at least two visual GCs

Application of zebrafish oculomotor behavior to model human disorders

To ensure high acuity vision, eye movements have to be controlled with astonishing precision by the oculomotor system. Many human diseases can lead to abnormal eye movements, typically of the involuntary oscillatory eye movements type called nystagmus. Such nystagmus can be congenital (infantile) or acquired later in life. Although the resulting eye movements are well characterized, there is only little information about the underlying etiology. This is in part owing to the lack of appropriate animal models. In this review article, we des­cribe how the zebrafish with its quick maturing visual system can be used to model oculomotor pathologies. We compare the characteristics and assessment of human and zebrafish eye movements. We describe the oculomotor properties of the zebrafish mutant belladonna, which has non-crossing optical fibers, and is a particularly informative model for human ocu­lomotor deficits. This mutant displays a reverse optokinetic response, spontaneous oscillations that closely mimic human congenital nystagmus and abnormal motor behavior linked to circular vectio

Whole-genome duplication in teleost fishes and its evolutionary consequences

Whole-genome duplication (WGD) events have shaped the history of many evolutionary lineages. One such duplication has been implicated in the evolution of teleost fishes, by far the most species-rich vertebrate clade. After initial controversy, there is now solid evidence that such event took place in the common ancestor of all extant teleosts. It is termed teleost-specific (TS) WGD. After WGD, duplicate genes have different fates. The most likely outcome is non-functionalization of one duplicate gene due to the lack of selective constraint on preserving both. Mechanisms that act on preservation of duplicates are subfunctionalization (partitioning of ancestral gene functions on the duplicates), neofunctionalization (assigning a novel function to one of the duplicates) and dosage selection (preserving genes to maintain dosage balance between interconnected components). Since the frequency of these mechanisms is influenced by the genes' properties, there are over-retained classes of genes, such as highly expressed ones and genes involved in neural function. The consequences of the TS-WGD, especially its impact on the massive radiation of teleosts, have been matter of controversial debate. It is evident that gene duplications are crucial for generating complexity and that WGDs provide large amounts of raw material for evolutionary adaptation and innovation. However, it is less clear whether the TS-WGD is directly linked to the evolutionary success of teleosts and their radiation. Recent studies let us conclude that TS-WGD has been important in generating teleost complexity, but that more recent ecological adaptations only marginally related to TS-WGD might have even contributed more to diversification. It is likely, however, that TS-WGD provided teleosts with diversification potential that can become effective much later, such as during phases of environmental change

Visual acuity in larval zebrafish: behavior and histology

BACKGROUND: Visual acuity, the ability of the visual system to distinguish two separate objects at a given angular distance, is influenced by the optical and neuronal properties of the visual system. Although many factors may contribute, the ultimate limit is photoreceptor spacing. In general, at least one unstimulated photoreceptor flanked by two stimulated ones is needed to perceive two objects as separate. This critical interval is also referred to as the Nyquist frequency and is according to the Shannon sampling theorem the highest spatial frequency where a pattern can be faithfully transmitted. We measured visual acuity in a behavioral experiment and compared the data to the physical limit given by photoreceptor spacing in zebrafish larvae. RESULTS: We determined visual acuity by using the optokinetic response (OKR), reflexive eye movements in response to whole field movements of the visual scene. By altering the spatial frequency we determined the visual acuity at approximately 0.16 cycles/degree (cpd) (minimum separable angle = 3.1 degrees ). On histological sections we measured the retinal magnification factor and the distance between double cones, that are thought to mediate motion perception. These measurements set the physical limit at 0.24 cpd (2.1 degrees ). CONCLUSION: The maximal spatial information as limited by photoreceptor spacing can not be fully utilized in a motion dependent visual behavior, arguing that the larval zebrafish visual system has not matured enough to optimally translate visual information into behavior. Nevertheless behavioral acuity is remarkable close to its maximal value, given the immature state of young zebrafish larvae

A robot-assisted acoustofluidic end effector

Liquid manipulation is the foundation of most laboratory processes. For macroscale liquid handling, both do-it-yourself and commercial robotic systems are available; however, for microscale, reagents are expensive and sample preparation is difficult. Over the last decade, lab-on-a-chip (LOC) systems have come to serve for microscale liquid manipulation; however, lacking automation andmulti-functionality. Despite their potential synergies, each has grown separately and no suitable interface yet exists to link macro-level robotics with micro-level LOC or microfluidic devices. Here, we present a robot-assisted acoustofluidic end effector (RAEE) system, comprising a robotic arm and an acoustofluidic end effector, that combines robotics and microfluidic functionalities.We further carried out fluid pumping, particle and zebrafish embryo trapping, and mobile mixing of complex viscous liquids. Finally, we pre-programmed the RAEE to perform automatedmixing of viscous liquids in well plates, illustrating its versatility for the automatic execution of chemical processes

Phylogenetic analysis of the vertebrate Excitatory/Neutral Amino Acid Transporter (SLC1/EAAT) family reveals lineage specific subfamilies

BACKGROUND: The composition and expression of vertebrate gene families is shaped by species specific gene loss in combination with a number of gene and genome duplication events (R1, R2 in all vertebrates, R3 in teleosts) and depends on the ecological and evolutionary context. In this study we analyzed the evolutionary history of the solute carrier 1 (SLC1) gene family. These genes are supposed to be under strong selective pressure (purifying selection) due to their important role in the timely removal of glutamate at the synapse. RESULTS: In a genomic survey where we manually annotated and analyzing sequences from more than 300 SLC1 genes (from more than 40 vertebrate species), we found evidence for an interesting evolutionary history of this gene family. While human and mouse genomes contain 7 SLC1 genes, in prototheria, sauropsida, and amphibia genomes up to 9 and in actinopterygii up to 13 SLC1 genes are present. While some of the additional slc1 genes in ray-finned fishes originated from R3, the increased number of SLC1 genes in prototheria, sauropsida, and amphibia genomes originates from specific genes retained in these lineages.Phylogenetic comparison and microsynteny analyses of the SLC1 genes indicate, that theria genomes evidently lost several SLC1 genes still present in the other lineage. The genes lost in theria group into two new subfamilies of the slc1 gene family which we named slc1a8/eaat6 and slc1a9/eaat7. CONCLUSIONS: The phylogeny of the SLC1/EAAT gene family demonstrates how multiple genome reorganization and duplication events can influence the number of active genes. Inactivation and preservation of specific SLC1 genes led to the complete loss of two subfamilies in extant theria, while other vertebrates have retained at least one member of two newly identified SLC1 subfamilies

Lack of the Sodium-Driven Chloride Bicarbonate Exchanger NCBE Impairs Visual Function in the Mouse Retina

Regulation of ion and pH homeostasis is essential for normal neuronal function. The sodium-driven chloride bicarbonate exchanger NCBE (Slc4a10), a member of the SLC4 family of bicarbonate transporters, uses the transmembrane gradient of sodium to drive cellular net uptake of bicarbonate and to extrude chloride, thereby modulating both intracellular pH (pHi) and chloride concentration ([Cl-]i) in neurons. Here we show that NCBE is strongly expressed in the retina. As GABAA receptors conduct both chloride and bicarbonate, we hypothesized that NCBE may be relevant for GABAergic transmission in the retina. Importantly, we found a differential expression of NCBE in bipolar cells: whereas NCBE was expressed on ON and OFF bipolar cell axon terminals, it only localized to dendrites of OFF bipolar cells. On these compartments, NCBE colocalized with the main neuronal chloride extruder KCC2, which renders GABA hyperpolarizing. NCBE was also expressed in starburst amacrine cells, but was absent from neurons known to depolarize in response to GABA, like horizontal cells. Mice lacking NCBE showed decreased visual acuity and contrast sensitivity in behavioral experiments and smaller b-wave amplitudes and longer latencies in electroretinograms. Ganglion cells from NCBE-deficient mice also showed altered temporal response properties. In summary, our data suggest that NCBE may serve to maintain intracellular chloride and bicarbonate concentration in retinal neurons. Consequently, lack of NCBE in the retina may result in changes in pHi regulation and chloride-dependent inhibition, leading to altered signal transmission and impaired visual function