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

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

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

The Binding Properties and Physiological Functions of Recoverin

Recoverin (Rcv) is a low molecular-weight, neuronal calcium sensor (NCS) primarily located in photoreceptor outer segments of the vertebrate retina. Calcium ions (Ca2+)-bound Rcv has been proposed to inhibit G-protein-coupled receptor kinase (GRKs) in darkness. During the light response, the Ca2+-free Rcv releases GRK, which in turn phosphorylates visual pigment, ultimately leading to the cessation of the visual transduction cascade. Technological advances over the last decade have contributed significantly to a deeper understanding of Rcv function. These include both biophysical and biochemical approaches that will be discussed in this review article. Furthermore, electrophysiological experiments uncovered additional functions of Rcv, such as regulation of the lifetime of Phosphodiesterase-Transducin complex. Recently, attention has been drawn to different roles in rod and cone photoreceptors.This review article focuses on Rcv binding properties to Ca2+, disc membrane and GRK, and its physiological functions in phototransduction and signal transmission

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 and multi-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 automated mixing of viscous liquids in well plates, illustrating its versatility for the automatic execution of chemical processes

Eumetazoan cryptochrome phylogeny and evolution

Cryptochromes (Crys) are light sensing receptors that are present in all eukaryotes. They mainly absorb light in the UV/blue spectrum. The extant Crys consist of two subfamilies, which are descendants of photolyases but are now involved in the regulation of circadian rhythms. So far, knowledge about the evolution, phylogeny, and expression of cry genes is still scarce. The inclusion of cry sequences from a wide range of bilaterian species allowed us to analyze their phylogeny in detail, identifying six major Cry subgroups. Selective gene inactivations and stabilizations in multiple chordate as well as arthropod lineages suggest several sub- and/or neofunctionalization events. An expression study performed in zebrafish, the model organism harboring the largest amount of crys, showed indeed only partially overlapping expression of paralogous mRNA, supporting gene sub- and/or neofunctionalization. Moreover, the daily cry expression in the adult zebrafish retina indicated varying oscillation patterns in different cell types. Our extensive phylogenetic analysis provides for the first time an overview of cry evolutionary history. Although several, especially parasitic or blind species, have lost all cry genes, crustaceans have retained up to three crys, teleosts possess up to seven, and tetrapods up to four crys. The broad and cyclic expression pattern of all cry transcripts in zebrafish retinal layers implies an involvement in retinal circadian processes and supports the hypothesis of several autonomous circadian clocks present in the vertebrate retina

Publisher Correction: The ciliopathy protein TALPID3/KIAA0586 acts upstream of Rab8 activation in zebrafish photoreceptor outer segment formation and maintenance

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper

Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming ciliary-directed vesicles, through organization of the vesicle fusion machinery at the periciliary membrane