Publications by authors named "Stephan C Neuhauss"

113 Publications

Color vision: Parsing spectral information for opponent color vision in the fish retina.

Curr Biol 2021 Dec;31(23):R1525-R1527

Department of Molecular Life Sciences, University of Zurich, Wintherthurerstrasse 190, CH - 8057 Zurich, Switzerland. Electronic address:

Environmental light carries spectral information, perceived as color. A new study in zebrafish shows how spectral information decoded by the cones' photoreceptors is transformed by retinal bipolar cells, adding a temporal component to the signal and establishing a third opponent axis for color vision.
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http://dx.doi.org/10.1016/j.cub.2021.10.016DOI Listing
December 2021

Loss of glutamate transporter eaat2a leads to aberrant neuronal excitability, recurrent epileptic seizures, and basal hypoactivity.

Glia 2022 Jan 30;70(1):196-214. Epub 2021 Oct 30.

Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

Astroglial excitatory amino acid transporter 2 (EAAT2, GLT-1, and SLC1A2) regulates the duration and extent of neuronal excitation by removing glutamate from the synaptic cleft. Hence, an impairment in EAAT2 function could lead to an imbalanced brain network excitability. Here, we investigated the functional alterations of neuronal and astroglial networks associated with the loss of function in the astroglia predominant eaat2a gene in zebrafish. We observed that eaat2a mutant zebrafish larvae display recurrent spontaneous and light-induced seizures in neurons and astroglia, which coincide with an abrupt increase in extracellular glutamate levels. In stark contrast to this hyperexcitability, basal neuronal and astroglial activity was surprisingly reduced in eaat2a mutant animals, which manifested in decreased overall locomotion. Our results reveal an essential and mechanistic contribution of EAAT2a in balancing brain excitability, and its direct link to epileptic seizures.
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http://dx.doi.org/10.1002/glia.24106DOI Listing
January 2022

DNA template strand segregation in developing zebrafish.

Cell Chem Biol 2021 11 29;28(11):1638-1647.e4. Epub 2021 Sep 29.

Department of Chemistry, University of Zurich, 8057 Zurich, Switzerland; Department of Pharmacology and Therapeutics, McGill University, Montreal, QC H3G 1Y6, Canada; Department of Chemistry, McGill University, Montreal, QC H3A 0B8, Canada. Electronic address:

Asymmetric inheritance of sister chromatids has long been predicted to be linked to discordant fates of daughter cells and even hypothesized to minimize accumulation of mutations in stem cells. Here, we use (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU), bromodeoxyuridine (BrdU), and light sheet microscopy to track embryonic DNA in whole zebrafish. Larval development results in rapid depletion of older DNA template strands from stem cell niches in the retina, brain, and intestine. Prolonged label retention occurs in quiescent progenitors that resume replication in later development. High-resolution microscopy reveals no evidence of asymmetric template strand segregation in >100 daughter cell pairs, making it improbable that asymmetric DNA segregation prevents mutational burden according to the immortal strand hypothesis in developing zebrafish.
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http://dx.doi.org/10.1016/j.chembiol.2021.09.001DOI Listing
November 2021

A Metabolic Landscape for Maintaining Retina Integrity and Function.

Front Mol Neurosci 2021 15;14:656000. Epub 2021 Apr 15.

Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

Neurons have high metabolic demands that are almost exclusively met by glucose supplied from the bloodstream. Glucose is utilized in complex metabolic interactions between neurons and glia cells, described by the astrocyte-neuron lactate shuttle (ANLS) hypothesis. The neural retina faces similar energy demands to the rest of the brain, with additional high anabolic needs to support continuous renewal of photoreceptor outer segments. This demand is met by a fascinating variation of the ANLS in which photoreceptors are the central part of a metabolic landscape, using glucose and supplying surrounding cells with metabolic intermediates. In this review we summarize recent evidence on how neurons, in particular photoreceptors, meet their energy and biosynthetic requirements by comprising a metabolic landscape of interdependent cells.
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http://dx.doi.org/10.3389/fnmol.2021.656000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8081888PMC
April 2021

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations.

Mol Ther 2021 08 23;29(8):2441-2455. Epub 2021 Apr 23.

Department of Otorhinolaryngology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525 GA Nijmegen, the Netherlands. Electronic address:

Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2a mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.
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http://dx.doi.org/10.1016/j.ymthe.2021.04.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8353187PMC
August 2021

Biochemistry and physiology of zebrafish photoreceptors.

Pflugers Arch 2021 09 17;473(9):1569-1585. Epub 2021 Feb 17.

Department of Molecular Life Sciences, University of Zurich, Winterthurerstrase 190, CH - 8057, Zürich, Switzerland.

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.
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http://dx.doi.org/10.1007/s00424-021-02528-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8370914PMC
September 2021

Fluorescent dATP for DNA Synthesis .

ACS Chem Biol 2020 11 27;15(11):2996-3003. Epub 2020 Oct 27.

Department of Chemistry, University of Zurich, Zurich CH-8006, Switzerland.

Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2'-deoxyadenosine-5'-triphosphate (dATP) derivatives of tetramethyl rhodamine ("TAMRA-dATP"), cyanine ("Cy3-dATP"), and boron-dipyrromethene ("BODIPY-dATP"). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode (). TAMRA-EdATP allows for unprecedented, real-time visualization of DNA replication and chromosome segregation .
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http://dx.doi.org/10.1021/acschembio.0c00654DOI Listing
November 2020

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

Genome Biol Evol 2020 11;12(11):2153-2167

Institute of Molecular Life Sciences, University of Zurich, Switzerland.

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.
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http://dx.doi.org/10.1093/gbe/evaa192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674705PMC
November 2020

Photoreceptor Degeneration Accompanies Vascular Changes in a Zebrafish Model of Diabetic Retinopathy.

Invest Ophthalmol Vis Sci 2020 02;61(2):43

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Purpose: Diabetic retinopathy (DR) is a leading cause of vision impairment and blindness worldwide in the working-age population, and the incidence is rising. Until now it has been difficult to define initiating events and disease progression at the molecular level, as available diabetic rodent models do not present the full spectrum of neural and vascular pathologies. Zebrafish harboring a homozygous mutation in the pancreatic transcription factor pdx1 were previously shown to display a diabetic phenotype from larval stages through adulthood. In this study, pdx1 mutants were examined for retinal vascular and neuronal pathology to demonstrate suitability of these fish for modeling DR.

Methods: Vessel morphology was examined in pdx1 mutant and control fish expressing the fli1a:EGFP transgene. We further characterized vascular and retinal phenotypes in mutants and controls using immunohistochemistry, histology, and electron microscopy. Retinal function was assessed using electroretinography.

Results: Pdx1 mutants exhibit clear vascular phenotypes at 2 months of age, and disease progression, including arterial vasculopenia, capillary tortuosity, and hypersprouting, could be detected at stages extending over more than 1 year. Neural-retinal pathologies are consistent with photoreceptor dysfunction and loss, but do not progress to blindness.

Conclusions: This study highlights pdx1 mutant zebrafish as a valuable complement to rodent and other mammalian models of DR, in particular for research into the mechanistic interplay of diabetes with vascular and neuroretinal disease. They are furthermore suited for molecular studies to identify new targets for treatment of early as well as late DR.
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http://dx.doi.org/10.1167/iovs.61.2.43DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329949PMC
February 2020

Publisher Correction: Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.

Sci Rep 2020 Feb 19;10(1):3326. Epub 2020 Feb 19.

Institut Jacques Monod, UMR7592 CNRS-Université Paris Diderot, Sorbonne Paris Cité, F-75205, Paris, France.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-58959-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031218PMC
February 2020

Transgenic zebrafish modeling low-molecular-weight proteinuria and lysosomal storage diseases.

Kidney Int 2020 06 28;97(6):1150-1163. Epub 2019 Dec 28.

Institute of Physiology, University of Zurich, Zurich, Switzerland. Electronic address:

Epithelial cells lining the proximal tubule of the kidney reabsorb and metabolize most of the filtered low-molecular-weight proteins through receptor-mediated endocytosis and lysosomal processing. Congenital and acquired dysfunctions of the proximal tubule are consistently reflected by the inappropriate loss of solutes including low-molecular-weight proteins in the urine. The zebrafish pronephros shares individual functional segments with the human nephron, including lrp2a/megalin-dependent endocytic transport processes of the proximal tubule. Although the zebrafish has been used as a model organism for toxicological studies and drug discovery, there is no available assay that allows large-scale assessment of proximal tubule function in larval or adult stages. Here we establish a transgenic Tg(lfabp::½vdbp-mCherry) zebrafish line expressing in the liver the N-terminal region of vitamin D-binding protein coupled to the acid-insensitive, red monomeric fluorescent protein mCherry (½vdbp-mCherry). This low-molecular-weight protein construct is secreted into the bloodstream, filtered through the glomerulus, reabsorbed by receptor-mediated endocytosis and processed in the lysosomes of proximal tubule cells of the fish. Thus, our proof-of-concept studies using zebrafish larvae knockout for lrp2a and clcn7 or exposed to known nephrotoxins (gentamicin and cisplatin) demonstrate that this transgenic line is useful to monitor low-molecular-weight proteinuria and lysosomal processing. This represents a powerful new model organism for drug screening and studies of nephrotoxicity.
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http://dx.doi.org/10.1016/j.kint.2019.11.016DOI Listing
June 2020

Author Correction: Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.

Sci Rep 2020 Jan 15;10(1):756. Epub 2020 Jan 15.

Institut Jacques Monod, UMR7592 CNRS-Université Paris Diderot, Sorbonne Paris Cité, F-75205, Paris, France.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-57829-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960163PMC
January 2020

A New Zebrafish Model for CACNA2D4-Dysfunction.

Invest Ophthalmol Vis Sci 2019 12;60(15):5124-5135

Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

Purpose: Mutations in CACNA2D4, encoding the α2δ4 subunit of retinal voltage-gated calcium channels (Cav), cause a rare type of retinal dysfunction in human, mainly affecting cone vision. Here, we investigate the role of CACNA2D4 in targeting of Cav, its influence on cone-mediated signal transmission, and the cellular and subcellular changes upon loss of α2δ4 by exploiting the advantages of the cone-dominant zebrafish as model system.

Methods: We identified two zebrafish CACNA2D4 paralogs (cacna2d4a and cacna2d4b), analyzed their expression by RNA in situ hybridization and introduced truncating frameshift mutations through CRISPR/Cas9-mediated mutagenesis. We analyzed retinal function and morphology of the single and double mutant lines by electroretinography, immunohistochemistry, light- and electron microscopy.

Results: Knockout of cacna2d4b reduces the expression of Cacna1fa, the pore-forming subunit of retinal Cav1.4, whereas loss of cacna2d4a did not. Only knockout of both paralogs impaired cone-mediated ERG b-wave amplitude. The number of "floating" ribbons is increased in double-KO, while retinal morphology and expression of postsynaptic mGluR6b remain largely unaffected. Both Cacna1fa and Ribeyeb show ectopic punctate expression in cacna2d4b-KO and double-KO photoreceptors.

Conclusions: We find that increasing the expression of Cav at the synaptic membrane is an evolutionarily conserved function of Cacna2d4b. Yet, since both paralogs participate in cone synaptic transmission, we propose partial subfunctionalization in zebrafish. Similar to human patients, our double KO zebrafish model shows mild cone dysfunction, which was not associated with signs of retinal degeneration. Therefore, cacna2d4-KO zebrafish is a suitable model to study the pathophysiological mechanisms underlying CACNA2D4 dysfunction in human.
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http://dx.doi.org/10.1167/iovs.19-26759DOI Listing
December 2019

Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.

Sci Rep 2019 03 18;9(1):4750. Epub 2019 Mar 18.

Institut Jacques Monod, UMR7592 CNRS-Université Paris Diderot, Sorbonne Paris Cité, F-75205, Paris, France.

Although structural nuclear pore proteins (nucleoporins) are seemingly required in every cell type to assemble a functional nuclear transport machinery, mutations or deregulation of a subset of them have been associated with specific human hereditary diseases. In particular, previous genetic studies of patients with nephrotic syndrome identified mutations in Nup107 that impaired the expression or the localization of its direct partner at nuclear pores, Nup133. In the present study, we characterized the zebrafish nup133 orthologous gene and its expression pattern during larval development. Using a morpholino-mediated gene knockdown, we show that partial depletion of Nup133 in zebrafish larvae leads to the formation of kidney cysts, a phenotype that can be rescued by co-injection of wild type mRNA. Analysis of different markers for tubular and glomerular development shows that the overall kidney development is not affected by nup133 knockdown. Likewise, no gross defect in nuclear pore complex assembly was observed in these nup133 morphants. On the other hand, nup133 downregulation results in proteinuria and moderate foot process effacement, mimicking some of the abnormalities typically featured by patients with nephrotic syndrome. These data indicate that nup133 is a new gene required for proper glomerular structure and function in zebrafish.
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http://dx.doi.org/10.1038/s41598-019-41202-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426968PMC
March 2019

The Binding Properties and Physiological Functions of Recoverin.

Front Mol Neurosci 2018 20;11:473. Epub 2018 Dec 20.

Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

Recoverin (Rcv) is a low molecular-weight, neuronal calcium sensor (NCS) primarily located in photoreceptor outer segments of the vertebrate retina. Calcium ions (Ca)-bound Rcv has been proposed to inhibit G-protein-coupled receptor kinase (GRKs) in darkness. During the light response, the Ca-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 Ca, disc membrane and GRK, and its physiological functions in phototransduction and signal transmission.
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http://dx.doi.org/10.3389/fnmol.2018.00473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306944PMC
December 2018

Differential expression of PKCα and -β in the zebrafish retina.

Histochem Cell Biol 2019 Jun 2;151(6):521-530. Epub 2019 Jan 2.

Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.

The retina is a complex neural circuit, which processes and transmits visual information from light perceiving photoreceptors to projecting retinal ganglion cells. Much of the computational power of the retina rests on signal integrating interneurons, such as bipolar cells. Commercially available antibodies against bovine and human conventional protein kinase C (PKC) α and -β are frequently used as markers for retinal ON-bipolar cells in different species, despite the fact that it is not known which bipolar cell subtype(s) they actually label. In zebrafish (Danio rerio) five prkc genes (coding for PKC proteins) have been identified. Their expression has not been systematically determined. While prkcg is not expressed in retinal tissue, the other four prkc (prkcaa, prkcab, prkcba, prkcbb) transcripts were found in different parts of the inner nuclear layer and some as well in the retinal ganglion cell layer. Immunohistochemical analysis in adult zebrafish retina using fluorescent in situ hybridization and PKC antibodies showed an overlapping immunolabeling of ON-bipolar cells that are most likely of the BON s6 and BON s6L or RRod type. However, comparison of transcript expression with immunolabeling, implies that these antibodies are not specific for one single zebrafish conventional PKC, but rather detect a combination of PKC -α and -β variants.
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http://dx.doi.org/10.1007/s00418-018-1764-8DOI Listing
June 2019

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

Sci Rep 2018 Aug 17;8(1):12534. Epub 2018 Aug 17.

Institute for Molecular Life Sciences, University of Zurich, 8057, Zurich, Switzerland.

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.
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http://dx.doi.org/10.1038/s41598-018-30671-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6098120PMC
August 2018

Sensory Biology: How to Structure a Tailor-Made Retina.

Curr Biol 2018 07;28(13):R737-R739

Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, CH - 8057 Zurich, Switzerland. Electronic address:

A new study of the zebrafish retina using sophisticated imaging has revealed how anisotropic properties of the retina are closely matched to the statistics of the natural visual world that the fish experiences.
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http://dx.doi.org/10.1016/j.cub.2018.05.020DOI Listing
July 2018

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

Sci Rep 2018 02 2;8(1):2211. Epub 2018 Feb 2.

Institute for Molecular Life Sciences, University of Zurich, 8057, Zurich, Switzerland.

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in signal transduction. Cilia are anchored inside the cell through basal bodies (BBs), modified centrioles also acting as microtubule-organization centers. Photoreceptors (PRs) are sensory neurons, whose primary cilium forms a highly specialized compartment called the outer segment (OS) responsible for sensing incoming light. Thus, ciliopathies often present with retinal degeneration. Mutations in KIAA0586/TALPID3 (TA3) cause Joubert syndrome, in which 30% of affected individuals develop retinal involvement. To elucidate the function of TALPID3 in PRs, we studied talpid3 zebrafish mutants and identified a progressive retinal degeneration phenotype. The majority of PRs lack OS development due to defects in BB positioning and docking at the apical cell surface. Intracellular accumulation of the photopigment opsin leads to PR cell death of moderate severity. Electroretinograms demonstrate severe visual impairement. A small subset of PRs display normally docked BBs and extended OSs through rescue by maternally-deposited Talpid3. While localization of the small GTPase Rab8a, which plays an important role in BB docking, appears unaffected in talpid3-/- PRs, overexpression of constitutively active Rab8a rescues OS formation, indicating that the role of Ta3 in early ciliogenesis lies upstream of Rab8a activation in PRs.
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http://dx.doi.org/10.1038/s41598-018-20489-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797153PMC
February 2018

Phylogeny and distribution of protein kinase C variants in the zebrafish.

J Comp Neurol 2018 05 1;526(7):1097-1109. Epub 2018 Feb 1.

University of Zurich, Institute of Molecular Life Sciences, Neuroscience Center Zurich and Center for Integrative Human Physiology, Zurich, CH-8057, Switzerland.

Conventional protein kinases-consisting of α, β, and γ family members-play key roles in numerous signal transduction events. Phylogenetic analysis demonstrated the existence of five prkcs (the genes representing PKCs) in zebrafish, two paralogous forms of prkca and prkcb and one prkcg variant. mRNA expression analysis showed distinct, mainly nervous system specific expression, for all five prkc genes. For prkca and prkcb paralogs prominent expression can be seen in the telencephalon, in diencephalic regions such as the habenula or the optic tectum, in hypothalamic areas and in distinct cerebellar structures. Each transcript is additionally expressed in distinct areas: prkcaa is highly abundant in cranial sensory ganglia and in dorsal neurons of the hindbrain and the spinal cord, prkcab is strongly expressed in additional cerebellar regions, prkcba shows expression in the pectoral fin, the otic vesicle and in the proximal convoluted tubule of the kidney, and prkcbb shows prominent expression in different hypothalamic areas. Expression of prkcg is most striking in the cerebellum. As zebrafish PKCs are expressed in structures that are equivalent to mammals, the zebrafish model is well suited to study evolutionary conserved functions of PKCs in development and disease.
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http://dx.doi.org/10.1002/cne.24395DOI Listing
May 2018

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.

PLoS Genet 2017 12 27;13(12):e1007150. Epub 2017 Dec 27.

Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

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.
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http://dx.doi.org/10.1371/journal.pgen.1007150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760100PMC
December 2017

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina.

J Vis Exp 2017 11 10(129). Epub 2017 Nov 10.

Center for Microscopy and Image Analysis, University of Zurich.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.
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http://dx.doi.org/10.3791/56113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755354PMC
November 2017

Genetic approaches to retinal research in zebrafish.

J Neurogenet 2017 09 5;31(3):70-87. Epub 2017 Jul 5.

a Institute of Molecular Life Sciences , University of Zurich , Zurich , Switzerland.

The zebrafish (Danio rerio) possesses a vertebrate-type retina that is extraordinarily conserved in evolution. This well-organized and anatomically easily accessible part of the central nervous system has been widely investigated in zebrafish, promoting general understanding of retinal development, morphology, function and associated diseases. Over the recent years, genome and protein engineering as well as imaging techniques have experienced revolutionary advances and innovations, creating new possibilities and methods to study zebrafish development and function. In this review, we focus on some of these emerging technologies and how they may impact retinal research in the future. We place an emphasis on genetic techniques, such as transgenic approaches and the revolutionizing new possibilities in genome editing.
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http://dx.doi.org/10.1080/01677063.2017.1343316DOI Listing
September 2017

Comparative transcriptomic analysis identifies evolutionarily conserved gene products in the vertebrate renal distal convoluted tubule.

Pflugers Arch 2017 Aug 27;469(7-8):859-867. Epub 2017 Jun 27.

Institute of Anatomy, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.

Understanding the molecular basis of the complex regulatory networks controlling renal ion transports is of major physiological and clinical importance. In this study, we aimed to identify evolutionarily conserved critical players in the function of the renal distal convoluted tubule (DCT) by a comparative transcriptomic approach. We generated a transgenic zebrafish line with expression of the red fluorescent mCherry protein under the control of the zebrafish DCT-specific promoter of the thiazide-sensitive NaCl cotransporter (NCC). The mCherry expression was then used to isolate from the zebrafish mesonephric kidneys the distal late (DL) segments, the equivalent of the mammalian DCT, for subsequent RNA-seq analysis. We next compared this zebrafish DL transcriptome to the previously established mouse DCT transcriptome and identified a subset of gene products significantly enriched in both the teleost DL and the mammalian DCT, including SLCs and nuclear transcription factors. Surprisingly, several of the previously described regulators of NCC (e.g., SPAK, KLHL3, ppp1r1a) in the mouse were not found enriched in the zebrafish DL. Nevertheless, the zebrafish DL expressed enriched levels of related homologues. Functional knockdown of one of these genes, ppp1r1b, reduced the phosphorylation of NCC in the zebrafish pronephros, similar to what was seen previously in knockout mice for its homologue, Ppp1r1a. The present work is the first report on global gene expression profiling in a specific nephron portion of the zebrafish kidney, an increasingly used model system for kidney research. Our study suggests that comparative analysis of gene expression between phylogenetically distant species may be an effective approach to identify novel regulators of renal function.
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http://dx.doi.org/10.1007/s00424-017-2009-8DOI Listing
August 2017

Mutations in ARMC9, which Encodes a Basal Body Protein, Cause Joubert Syndrome in Humans and Ciliopathy Phenotypes in Zebrafish.

Am J Hum Genet 2017 Jul 15;101(1):23-36. Epub 2017 Jun 15.

Department of Pediatrics, University of Washington, Seattle, WA 98195, USA; Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA 98101, USA. Electronic address:

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by hypotonia, ataxia, abnormal eye movements, and variable cognitive impairment. It is defined by a distinctive brain malformation known as the "molar tooth sign" on axial MRI. Subsets of affected individuals have malformations such as coloboma, polydactyly, and encephalocele, as well as progressive retinal dystrophy, fibrocystic kidney disease, and liver fibrosis. More than 35 genes have been associated with JS, but in a subset of families the genetic cause remains unknown. All of the gene products localize in and around the primary cilium, making JS a canonical ciliopathy. Ciliopathies are unified by their overlapping clinical features and underlying mechanisms involving ciliary dysfunction. In this work, we identify biallelic rare, predicted-deleterious ARMC9 variants (stop-gain, missense, splice-site, and single-exon deletion) in 11 individuals with JS from 8 families, accounting for approximately 1% of the disorder. The associated phenotypes range from isolated neurological involvement to JS with retinal dystrophy, additional brain abnormalities (e.g., heterotopia, Dandy-Walker malformation), pituitary insufficiency, and/or synpolydactyly. We show that ARMC9 localizes to the basal body of the cilium and is upregulated during ciliogenesis. Typical ciliopathy phenotypes (curved body shape, retinal dystrophy, coloboma, and decreased cilia) in a CRISPR/Cas9-engineered zebrafish mutant model provide additional support for ARMC9 as a ciliopathy-associated gene. Identifying ARMC9 mutations as a cause of JS takes us one step closer to a full genetic understanding of this important disorder and enables future functional work to define the central biological mechanisms underlying JS and other ciliopathies.
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http://dx.doi.org/10.1016/j.ajhg.2017.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501774PMC
July 2017

Shaping of Signal Transmission at the Photoreceptor Synapse by EAAT2 Glutamate Transporters.

eNeuro 2017 May-Jun;4(3). Epub 2017 Jun 12.

Institute of Molecular Life Sciences, University of Zurich, Zurich, CH-8057, Switzerland.

Photoreceptor ribbon synapses tonically release glutamate. To ensure efficient signal transmission and prevent glutamate toxicity, a highly efficient glutamate removal system provided by members of the SLC1 gene family is required. By using a combination of biophysical and studies, we elucidate the role of excitatory amino acid transporter 2 (EAAT2) proteins in synaptic glutamate homeostasis at the zebrafish photoreceptor synapse. The main glutamate sink is provided by the glial EAAT2a, reflected by reduced electroretinographic responses in EAAT2a-depleted larvae. EAAT2b is located on the tips of cone pedicles and contributes little to glutamate reuptake. However, this transporter displays both a large chloride conductance and leak current, being important in stabilizing the cone resting potential. This work demonstrates not only how proteins originating from the same gene family can complement each other's expression profiles and biophysical properties, but also how presynaptic and glial transporters are coordinated to ensure efficient synaptic transmission at glutamatergic synapses of the central nervous system.
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http://dx.doi.org/10.1523/ENEURO.0339-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467398PMC
March 2018

Olfaction: How Fish Catch a Whiff.

Curr Biol 2017 01;27(2):R57-R58

University of Zurich, Institute of Molecular Life Sciences, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Electronic address:

An elegant new study shows that multiciliated cells in the noses of aquatic vertebrates generate flow fields that help odor detection and processing.
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http://dx.doi.org/10.1016/j.cub.2016.12.007DOI Listing
January 2017

A Zebrafish Model for Infection with the Obligate Intracellular Pathogen .

Front Microbiol 2016 18;7:1829. Epub 2016 Nov 18.

Vetsuisse Faculty, Institute for Veterinary Pathology, University of Zurich Zurich, Switzerland.

Obligate intracellular chlamydial bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum are important pathogens of terrestrial and marine vertebrates, yet many features of their pathogenesis and host specificity are still unknown. This is particularly true for families such as the which, in addition to epithelia, cellular targets for nearly all , can infect and replicate in macrophages, an important arm of the innate immune system or in their free-living amoebal counterparts. An ideal pathogen model system should include both host and pathogen, which led us to develop the first larval zebrafish model for chlamydial infections with . By varying the means and sites of application, epithelial cells of the swim bladder, endothelial cells of the vasculature and phagocytosing cells of the innate immune system became preferred targets for infection in zebrafish larvae. Through the use of transgenic zebrafish, we could observe recruitment of neutrophils to the infection site and demonstrate for the first time that is taken up and replicates in these phagocytic cells and not only in macrophages. Furthermore, we present evidence that myeloid differentiation factor 88 (MyD88) mediated signaling plays a role in the innate immune reaction to , eventually by Toll-like receptor (TLRs) recognition. Infected larvae with depleted levels of MyD88 showed a higher infection load and a lower survival rate compared to control fish. This work presents a new and potentially powerful non-mammalian experimental model to study the pathology of chlamydial virulence and opens up new possibilities for investigation of other members of the PVC superphylum.
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http://dx.doi.org/10.3389/fmicb.2016.01829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114312PMC
November 2016

Important role of endocannabinoid signaling in the development of functional vision and locomotion in zebrafish.

FASEB J 2016 12 13;30(12):4275-4288. Epub 2016 Sep 13.

Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy;

The developmental role of the endocannabinoid system still remains to be fully understood. Here, we report the presence of a complete endocannabinoid system during zebrafish development and show that the genes that code for enzymes that catalyze the anabolism and catabolism (mgll and dagla) of the endocannabinoid, 2-AG (2-arachidonoylglycerol), as well as 2-AG main receptor in the brain, cannabinoid receptor type 1, are coexpressed in defined regions of axonal growth. By using morpholino-induced transient knockdown of the zebrafish Daglα homolog and its pharmacologic rescue, we suggest that synthesis of 2-AG is implicated in the control of axon formation in the midbrain-hindbrain region and that animals that lack Daglα display abnormal physiological behaviors in tests that measure stereotyped movement and motion perception. Our results suggest that the well-established role for 2-AG in axonal outgrowth has implications for the control of vision and movement in zebrafish and, thus, is likely common to all vertebrates.-Martella, A., Sepe, R. M., Silvestri, C., Zang, J., Fasano, G., Carnevali, O., De Girolamo, P., Neuhauss, S. C. F., Sordino, P., Di Marzo, V. Important role of endocannabinoid signaling in the development of functional vision and locomotion in zebrafish.
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http://dx.doi.org/10.1096/fj.201600602RDOI Listing
December 2016
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