News
Recirculating aquaculture systems (RAS) are increasingly being used to grow fish, as intensive water reuse reduces water consumption and environmental impact. RAS use biofilters containing nitrogen-cycling microorganisms that remove ammonia from the aquaculture water. Knowledge of how RAS microbial communities relate to the fish-associated microbiome is limited, as is knowledge of fish-associated microbiota in general. Recently, nitrogen-cycling bacteria have been discovered in zebrafish and carp gills and shown to detoxify ammonia in a manner similar to the RAS biofilter. Researchers from Microbiology and Animal Physiology, working together in the Radboud Zebrafish facility, compared RAS water and biofilter microbiomes with fish-associated gut and gill microbial communities in laboratory RAS housing either zebrafish (Danio rerio) or common carp (Cyprinus carpio) using 16S rRNA gene amplicon sequencing.
3 July 2023
The paper "Generation and Characterization of a Zebrafish Model for ADGRV1-Associated Retinal Dysfunction Using CRISPR/Cas9 Genome Editing Technology" has been published in a Special Issue of Cells "CRISPR-Based Genome Editing in Translational Research".In the article, researchers working within our facility describe that worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the ADGRV1 gene, for which currently no treatment options exist. A model organism that mimics the human phenotype is essential to unravel the exact pathophysiological mechanism underlying ADGRV1-associated RP, and to evaluate future therapeutic strategies. The introduction of CRISPR/Cas-based genome editing technologies significantly improved the possibilities of generating mutant models in a time- and cost-effective manner. Zebrafish have been recognized as a suitable model to study Usher syndrome-associated retinal dysfunction. Using CRISPR/Cas9 technology we introduced a 4bp deletion in adgrv1 exon 9 (adgrv1rmc22). Immunohistochemical analysis showed that Adgrv1 was absent from the region of the photoreceptor connecting cilium in the adgrv1rmc22 zebrafish retina. Here, the absence of Adgrv1 also resulted in reduced levels of the USH2 complex members usherin and Whrnb, suggesting that Adgrv1 interacts with usherin and Whrnb in zebrafish photoreceptors. When comparing adgrv1rmc22 zebrafish with wild-type controls, we furthermore observed increased levels of aberrantly localized rhodopsin in the photoreceptor cell body, and decreased electroretinogram (ERG) B-wave amplitudes which indicate that the absence of Adgrv1 results in impaired retinal function. Based on these findings we present the adgrv1rmc22 zebrafish as the first ADGRV1 mutant model that displays an early retinal dysfunction. Moreover, the observed phenotypic changes can be used as quantifiable outcome measures when evaluating the efficacy of future novel therapeutic strategies for ADGRV1-associated RP.
16 June 2023
Loss-of-function mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). We previously presented skipping of USH2A exon 13 as a promising treatment paradigm for USH2A-associated RP. However, RP-associated mutations are often private, and evenly distributed along the USH2A gene. In order to broaden the group of patients that could benefit from therapeutic exon skipping strategies, we expanded our approach to other USH2A exons in which unique loss-of-function mutations have been reported by implementing a protein domain-oriented dual exon skipping strategy. We first generated zebrafish mutants carrying a genomic deletion of the orthologous exons of the frequently mutated human USH2A exons 30–31 or 39–40 using CRISPR-Cas9. Excision of these in-frame combinations of exons restored usherin expression in the zebrafish retina and rescued the photopigment mislocalization typically observed in ush2a mutants. To translate these findings into a future treatment in humans, we employed in vitro assays to identify and validate antisense oligonucleotides (ASOs) with a high potency for sequence-specific dual exon skipping. Together, the in vitro and in vivo data demonstrate protein domain-oriented ASO-induced dual exon skipping to be a highly promising treatment option for RP caused by mutations in USH2A.
16 June 2023
A joint initiative between researchers at RIBES and Radboudumc has led to new insights into the functions of a gene that in humans is implicated in cleft palate and thyroid dysgenesis. A zebrafish mutant was generated that has a disruption in the nuclear localization signal in the foxe1 gene. Skeletal development and thyroidogenesis in these mutants were investigated, focusing on embryonic and larval stages.
13 March 2023
Ammonia accumulation is a major challenge in intensive aquaculture, where fish are fed protein-rich diets in large rations, resulting in increased ammonia production when amino acids are metabolized as energy source. Ammonia is primarily excreted via the gills, which have been found to harbor nitrogen-cycle bacteria that convert ammonia into dinitrogen gas (N2) and therefore present a potential in situ detoxifying mechanism.A research team from Animal Physiology and Microbiology, in collaboration with Wageningen University, determined the impact of feeding strategies (demand-feeding and batch-feeding) with two dietary protein levels on growth, nitrogen excretion, and nitrogen metabolism in common carp (Cyprinus carpio, L.) in a 3-week feeding experiment. Demand-fed fish exhibited significantly higher growth rates, though with lower feed efficiency. When corrected for feed intake, nitrogen excretion was not impacted by feeding strategy or dietary protein, but demand-fed fish had significantly more nitrogen unaccounted for in the nitrogen balance and less retained nitrogen. N2 production of individual fish was measured in all experimental groups, and production rates were in the same order of magnitude as the amount of nitrogen unaccounted for, thus potentially explaining the missing nitrogen in the balance. N2 production by carp was also observed when groups of fish were kept in metabolic chambers. Demand feeding furthermore caused a significant increase in hepatic glutamate dehydrogenase activities, indicating elevated ammonia production. However, branchial ammonia transporter expression levels in these animals were stable or decreased. Together, the results suggest that feeding strategy impacts fish growth and nitrogen metabolism, and that conversion of ammonia to N2 by nitrogen cycle bacteria in the gills may explain the unaccounted nitrogen in the balance.
7 February 2023
Pharmacological targeting of FGFR1-4 kinase signaling causes severe craniofacial malformations in zebrafish larvae, whereas abrogation of FGFR2 kinase signaling alone does not induce craniofacial skeletal abnormalities. These findings were recently published by researchers from Dentistry (Radboudumc) and Animal Ecology and Physiology (Radboud University). The results will further the understanding of the role of FGFRs in the etiology of craniofacial malformations.
17 December 2022
In the last decade, predictions of impacts of climate change on biodiversity, and how to model changes in species distributions have gained relatively more attention than the importance of understanding physiological principles of an organism in predicting its response to climate change. Therefore, we recently launched a PhD research project into the combined effects of climate change factors (thermal stress, hypoxia, acidification) on (skeletal) development in early fish developmental stages. The investigation will increase our insights in the effects and interactions of climate change-induced environmental changes on fish development and vulnerability.
12 December 2022
The Radboud Zebrafish Facility has two new zebrafish systems that together provide space for more than 250 tanks. They are the most modern, recirculating housing systems, equipped with light blue polycarbonate tanks to prevent algae growth. The systems are also equipped with mechanical, chemical and biological filtration, UV disinfection and buffering. In this way, less stress for the animals and better fish welfare is guaranteed.
16 August 2022
The molecular mechanisms of retinal degeneration due to compromised formation and function of the USH2-associated protein complex remain elusive. In a recent publication, researchers joining in the Radboud Zebrafish Facility took another step to unravel this pathogenic mechanism. They have isolated and characterized whirlin-associated protein complexes from zebrafish photoreceptor cells. They generated transgenic zebrafish that express Strep/FLAG-tagged Whrna, a zebrafish ortholog of human whirlin, under the control of a photoreceptor-specific promoter.
8 July 2022
Since linkage of human RAB28 mutations to inherited blindness and cone-rod dystrophy, studies have focused on Rab28 function in the retina. However, there remains a vast gap in understanding of the protein networks regulated by Rab28. Researchers from, among others, the zebrafish facility endeavored to bridge that void, to shed more light on Rab28 function and Rab28-CRD disease mechanisms. Thie study uncovered that Rab28 is required from the larval zebrafish stage for the burst of RPE phagocytosis, of the photoreceptor outer segments at both dawn and dusk peaks. They further investigated the regulation of OSP in the zebrafish model and the role of Rab28. Using a gene complementation approach, it was found that Rab28 plays an important role in the cone photoreceptors, as cone-specific expression of Rab28 was sufficient to restore the OSP defect in the knockout model. Proteomic analysis by mass spectrometry demonstrates loss of Rab28 effects the expression profile of several known regulators of OSP and identified potential novel regulators of the OSP process. rab28 KO can result in long-term physiological effects on the retina, as retinoid and bisretinoid analysis on therab28 KO revealed altered levels of visual cycle components, as well as an increase in the toxic vitamin A compound A2PE.
9 June 2022