Mutant Zebrafish Model Research Articles

The Reissner Fiber Is Highly Dynamic In Vivo and Controls Morphogenesis of the Spine

Cerebrospinal fluid (CSF) physiology is important for the development and homeostasis of the central nervous system, and its disruption has been linked to scoliosis in zebrafish [1, 2]. Suspended in the CSF is an extracellular structure called the Reissner fiber, which extends from the brain through the central canal of the spinal cord. Zebrafish scospondin-null mutants are unable to assemble a Reissner fiber and fail to form a straight body axis during embryonic development [3]. Here, we describe hypomorphic missense mutations of scospondin, which allow Reissner fiber assembly and extension of a straight axis. However, during larval development, these mutants display progressive Reissner fiber disassembly, which is concomitant with the emergence of axial curvatures and scoliosis in adult animals. Using a scospondin-GFP knockin zebrafish line, we demonstrate several dynamic properties of the Reissner fiber invivo, including embryonic fiber assembly, the continuous rostral to caudal movement of the fiber within the brain and central canal, and subcommissural organ (SCO)-spondin-GFP protein secretion from the floor plate to merge with the fiber. Finally, we show that disassembly of the Reissner fiber is also associated with the progression of axial curvatures in distinct scoliosis mutant zebrafish models. Together, these data demonstrate a critical role for the Reissner fiber for the maintenance of a straight body axis and spine morphogenesis in adult zebrafish. Our study establishes a framework for future investigations to address the cellular effectors responsible for Reissner-fiber-dependent regulation of axial morphology. VIDEO ABSTRACT.

A252 FUNCTIONAL ANALYSIS OF GENETIC AETIOLOGIES IN A DOWNSTREAM OF TYROSINE KINASE (DOK) PROTEIN IN THE PATHOGENESIS OF PEDIATRIC INFLAMMATORY BOWEL DISEASE (IBD)

Abstract Background IBD is a form of chronic inflammatory disorder of the gastrointestinal tract that arises due to genetic, environmental, immunological and microbial factors. The precise pathological mechanisms remain elusive. It is thought that the onset of pediatric IBD can largely be attributed to genetics. Muise lab, at SickKids, regularly screens children at the SickKids IBD clinic and through an international consortium to find possible genetic links to the disease. We report a patient at SickKids with biallelic mutations in DOK4 who has severe Crohn’s Disease along with other inflammatory conditions. Downstream of kinase (DOK) proteins are a family of adaptor molecules that serve as scaffolding proteins important in regulating cell signaling, especially in T cells. DOK4 has been shown to have negative regulatory effects on T cell activation but is also expressed across various other tissues where its function is yet to be determined. We predict that these mutations are causing immune cell dysregulation, which may be contributing to the patients IBD. Aims Through this study, we aim to enhance our understanding of the pathobiological mechanism of novel mutations in DOK4. Methods We have established T cell lines, expressing wild type and mutated DOK4, which will be used to perform functional tests, such as localization analysis through immunofluorescence and cytokine profiling, to check for T cell function. We have patient derived organoids, which will be used to assess changes in gut morphology using imaging techniques. We will also generate mutant zebrafish model that will be used to determine the susceptibility to colitis related to this mutation, disease progression and gut peristalsis using live imaging technology. Results Preliminary data shows variation in expression of the protein within patient derived peripheral blood mononuclear cells (PBMCs) compared to a healthy donor. Conclusions With this study, we hope to identify new therapeutic targets for patients with DOK4 mutations. Funding Agencies CIHRThe Leona M. and Harry B. Helmsley Charitable Trust

Modeling ZNF408-Associated FEVR in Zebrafish Results in Abnormal Retinal Vasculature.

PurposeFamilial exudative vitreoretinopathy (FEVR) is an inherited retinal disease in which the retinal vasculature is affected. Patients with FEVR typically lack or have abnormal vasculature in the peripheral retina, the outcome of which can range from mild visual impairment to complete blindness. A missense mutation (p.His455Tyr) in ZNF408 was identified in an autosomal dominant FEVR family. Little, however, is known about the molecular role of ZNF408 and how its defect leads to the clinical features of FEVR.MethodsUsing CRISPR/Cas9 technology, two homozygous mutant zebrafish models with truncated znf408 were generated, as well as one heterozygous and one homozygous missense znf408 model in which the human p.His455Tyr mutation is mimicked.ResultsIntriguingly, all three znf408-mutant zebrafish strains demonstrated progressive retinal vascular pathology, initially characterized by a deficient hyaloid vessel development at 5 days postfertilization (dpf) leading to vascular insufficiency in the retina. The generation of stable mutant lines allowed long-term follow up studies, which showed ectopic retinal vascular hyper-sprouting at 90 dpf and extensive vascular leakage at 180 dpf.ConclusionsTogether, our data demonstrate an important role for znf408 in the development and maintenance of the vascular system within the retina.

Genotypic and Phenotypic Variables Affect Meiotic Cell Cycle Progression, Tumor Ploidy, and Cancer-Associated Mortality in a brca2-Mutant Zebrafish Model.

Successful cell replication requires both cell cycle completion and accurate chromosomal segregation. The tumor suppressor BRCA2 is positioned to influence both of these outcomes, and thereby influence genomic integrity, during meiotic and mitotic cell cycles. Accordingly, mutations in BRCA2 induce chromosomal abnormalities and disrupt cell cycle progression in both germ cells and somatic cells. Despite these findings, aneuploidy is not more prevalent in BRCA2-associated versus non-BRCA2-associated human cancers. More puzzlingly, diploidy in BRCA2-associated cancers is a negative prognostic factor, unlike non-BRCA2-associated cancers and many other human cancers. We used a brca2-mutant/tp53-mutant cancer-prone zebrafish model to explore the impact of BRCA2 mutation on cell cycle progression, ploidy, and cancer-associated mortality by performing DNA content/cell cycle analysis on zebrafish germ cells, somatic cells, and cancer cells. First, we determined that combined brca2/tp53 mutations uniquely disrupt meiotic progression. Second, we determined that sex significantly influences ploidy outcome in zebrafish cancers. Third, we determined that brca2 mutation and female sex each significantly reduce survival time in cancer-bearing zebrafish. Finally, we provide evidence to support a link between BRCA2 mutation, tumor diploidy, and poor survival outcome. These outcomes underscore the utility of this model for studying BRCA2-associated genomic aberrations in normal and cancer cells.

Zebrafish studies identify serotonin receptors mediating antiepileptic activity in Dravet syndrome.

Dravet syndrome is a life-threatening early-onset epilepsy not well controlled by antiepileptic drugs. Drugs that modulate serotonin (5-HT) signalling, including clemizole, locaserin, trazodone and fenfluramine, have recently emerged as potential treatment options for Dravet syndrome. To investigate the serotonin receptors that could moderate this antiepileptic activity, we designed and synthesized 28 novel analogues of clemizole, obtained receptor binding affinity profiles, and performed in vivo screening in a scn1lab mutant zebrafish (Danio rerio) model which recapitulates critical clinical features of Dravet syndrome. We discovered three clemizole analogues with 5-HT receptor binding that exert powerful antiepileptic activity. Based on structure-activity relationships and medicinal chemistry-based analysis, we then screened an additional set of known 5-HT receptor specific drug candidates. Integrating our in vitro and in vivo data implicates 5-HT2B receptors as a critical mediator in the mechanism of seizure suppression observed in Dravet syndrome patients treated with 5-HT modulating drugs.

Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish.

Mutations in eyes shut homolog (EYS), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive retinal disorder. Due to the absence of EYS in several rodent species and its retina-specific expression, still little is known about the exact function of EYS and the pathogenic mechanism underlying EYS-associated RP. We characterized eys in zebrafish, by RT-PCR analysis on zebrafish eye-derived RNA, which led to the identification of a 8,715 nucleotide coding sequence that is divided over 46 exons. The transcript is predicted to encode a 2,905-aa protein that contains 39 EGF-like domains and five laminin A G-like domains, which overall shows 33% identity with human EYS. To study the function of EYS, we generated a stable eysrmc101/rmc101 mutant zebrafish model using CRISPR/Cas9 technology. The introduced lesion is predicted to result in premature termination of protein synthesis and lead to loss of Eys function. Immunohistochemistry on retinal sections revealed that Eys localizes at the region of the connecting cilium and that both rhodopsin and cone transducin are mislocalized in the absence of Eys. Electroretinogram recordings showed diminished b-wave amplitudes in eysrmc101/rmc101 zebrafish (5 dpf) compared to age- and strain-matched wild-type larvae. In addition, decreased locomotor activity in response to light stimuli was observed in eys mutant larvae. Altogether, our study shows that absence of Eys leads to a disorganized retinal architecture and causes visual dysfunction in zebrafish.

Generation and Validation of a Complete Knockout Model of abcc6a in Zebrafish.

Pseudoxanthoma elasticum is an ectopic mineralization disease due to biallelic ABCC6 mutations. As no treatment options are currently available, a reliable zebrafish model is invaluable for high throughput compound screening. However, data from previously reported knockdown and mutant zebrafish models for abcc6a, the functional orthologue of ABCC6, showed phenotypic discrepancies. To address this, we developed a complete abcc6a knockout model using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 and compared its phenotype to that of a mutant model (Sa963) and a splice junction morpholino model. Our data showed that abcc6a is not required for embryonic survival, but rather that it has an essential role in controlling mineralization. The three models developed very similar hypermineralization of spine and ribs starting embryonically and progressing in adulthood with development of scoliosis. Our results indicate a direct relation between loss of abcc6a expression and dysregulated osteogenesis. As such, our models recapitulate part of the human phenotype in which ectopic mineralization and pro-osteogenic signaling have been reported. Because of its reproducibility in three models and its ease of quantification, we consider this phenotype to be unequivocally the result of abcc6 deficiency and, as such, an excellent readout for drug screening purposes and multiplex mutagene analysis.

C2orf71a/pcare1 is important for photoreceptor outer segment morphogenesis and visual function in zebrafish

Mutations in C2orf71 are causative for autosomal recessive retinitis pigmentosa and occasionally cone-rod dystrophy. We have recently discovered that the protein encoded by this gene is important for modulation of the ciliary membrane through the recruitment of an actin assembly module, and have therefore renamed the gene to PCARE (photoreceptor cilium actin regulator). Here, we report on the identification of two copies of the c2orf71/pcare gene in zebrafish, pcare1 and pcare2. To study the role of the gene most similar to human PCARE, pcare1, we have generated a stable pcare1 mutant zebrafish model (designated pcare1rmc100/rmc100) in which the coding sequence was disrupted using CRISPR/Cas9 technology. Retinas of both embryonic (5 dpf) and adult (6 mpf) pcare1rmc100/rmc100 zebrafish display a clear disorganization of photoreceptor outer segments, resembling the phenotype observed in Pcare−/− mice. Optokinetic response and visual motor response measurements indicated visual impairment in pcare1rmc100/rmc100 zebrafish larvae at 5 dpf. In addition, electroretinogram measurements showed decreased b-wave amplitudes in pcare1rmc100/rmc100 zebrafish as compared to age- and strain-matched wild-type larvae, indicating a defect in the transretinal current. Altogether, our data show that lack of pcare1 causes a retinal phenotype in zebrafish and indicate that the function of the PCARE gene is conserved across species.

CRISPR/Cas9-induced shank3b mutant zebrafish display autism-like behaviors

BackgroundHuman genetic and genomic studies have supported a strong causal role of SHANK3 deficiency in autism spectrum disorder (ASD). However, the molecular mechanism underlying SHANK3 deficiency resulting in ASD is not fully understood. Recently, the zebrafish has become an attractive organism to model ASD because of its high efficiency of genetic manipulation and robust behavioral phenotypes. The orthologous gene to human SHANK3 is duplicated in the zebrafish genome and has two homologs, shank3a and shank3b. Previous studies have reported shank3 morphants in zebrafish using the morpholino method. Here, we report the generation and characterization of shank3b mutant zebrafish in larval and adult stages using the CRISPR/Cas9 genome editing technique.MethodsCRISPR/Cas9 was applied to generate a shank3b loss-of-function mutation (shank3b−/−) in zebrafish. A series of morphological measurements, behavioral tests, and molecular analyses were performed to systematically characterize the behavioral and molecular changes in shank3b mutant zebrafish.Resultsshank3b−/− zebrafish exhibited abnormal morphology in early development. They showed reduced locomotor activity both as larvae and adults, reduced social interaction and time spent near conspecifics, and significant repetitive swimming behaviors. Additionally, the levels of both postsynaptic homer1 and presynaptic synaptophysin were significantly reduced in the adult brain of shank3b-deficient zebrafish.ConclusionsWe generated the first inheritable shank3b mutant zebrafish model using CRISPR/Cas9 gene editing approach. shank3b−/− zebrafish displayed robust autism-like behaviors and altered levels of the synaptic proteins homer1 and synaptophysin. The versatility of zebrafish as a model for studying neurodevelopment and conducting drug screening will likely have a significant contribution to future studies of human SHANK3 function and ASD.

CFTR mutation enhances Dishevelled degradation and results in impairment of Wnt-dependent hematopoiesis

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) with a multitude of clinical manifestations. Some CF patients develop clinically significant anemia, suggesting that CFTR may regulate hematopoiesis. Here, we report that cftr mutant zebrafish model exhibits primitive and definitive hematopoietic defects with impaired Wnt signaling. Cftr is found to interact, via its PDZ-binding domain (PDZBD), with Dishevelled (Dvl), a key component of Wnt signaling required for hematopoietic progenitor specification, thus protecting Dvl from Dapper1 (Dpr1)-induced lysosomal degradation. Defective hematopoiesis and impaired Wnt signaling in cftr mutant can be rescued by overexpression of wild-type or channel function-defective G551D mutant CFTR with an intact PDZBD, but not Cftr with mutations in the PDZBD. Analysis of human database (http://r2.amc.nl) shows that CFTR is positively correlated with DVL2 and Wnt-related hematopoietic factors in human blood system. The results reveal a previously unrecognized role of CFTR, which is independent of its channel function, in regulating DVL degradation and thus Wnt signaling required for hematopoiesis in both zebrafish and humans, providing an explanation for the anemic phenotype of CF patients.

Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction

The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.

Transcript analysis of natural killer (NK) cell specific genes in the liver, kidney and spleen tissues of rag1 −/− mutant zebrafish in response to in vivo administration of TLR ligands

Abstract T and B cells mediate specific protective immunity. NK cells can also mediate a memory response to murine cytomegalovirus in T and B cell deficient rag2−/− mice. T and B cell deficient rag1−/− mutant zebrafish mediate protective immunity to intracellular bacteria. NK cells have not been characterized in zebrafish due to a lack of tools to target specific markers. The aim of this study was to evaluate NK cell tissue distribution and transcript response to TLR ligands in rag1−/− mutant zebrafish. We accomplished this by determining the level of expression of ifnγ, t-bet, nitr9, and NK lysin (nkl) a, b, c and d transcripts following exposure to β glucan, Poly I:C, R-848 and VTX-2337. Expression levels were normalized to the basal level of expression and the fold changes were compared to that of endotoxin free PBS (carrier control) injected group. R848 and VTX-2337 exposure changed the gene expression profile in a tissue specific manner. Nitr9 protein expression was analyzed by western blot and detectable levels were found in the liver. Nitr9 expression correlated with the expression of ifnγ in liver and kidney. In another experiment, fish were injected with TLR ligand when vaccinated, were only vaccinated, or not vaccinated. After bacteria challenge, fish that received β glucan or R848 when vaccinated had the highest survival. This suggests the TLR ligands have an adjuvant activity on NK cell protective responses. Our results of NK cell stimulation indirectly demonstrate the presence of NK cells in zebrafish and suggest a possible means of enhancing NK cell based immunity. These findings demonstrate the clinical significance of the rag1−/− mutant zebrafish model.

Establishing an LRP5 mutant zebrafish (D. Rerio) model of bone acquisition

Establishing an LRP5 mutant zebrafish (D. Rerio) model of bone acquisition

Small 6q16.1 Deletions Encompassing POU3F2 Cause Susceptibility to Obesity and Variable Developmental Delay with Intellectual Disability

Genetic studies of intellectual disability and identification of monogenic causes of obesity in humans have made immense contribution toward the understanding of the brain and control of body mass. The leptin > melanocortin > SIM1 pathway is dysregulated in multiple monogenic human obesity syndromes but its downstream targets are still unknown. In ten individuals from six families, with overlapping 6q16.1 deletions, we describe a disorder of variable developmental delay, intellectual disability, and susceptibility to obesity and hyperphagia. The 6q16.1 deletions segregated with the phenotype in multiplex families and were shown to be de novo in four families, and there was dramatic phenotypic overlap among affected individuals who were independently ascertained without bias from clinical features. Analysis of the deletions revealed a ∼350 kb critical region on chromosome 6q16.1 that encompasses a gene for proneuronal transcription factor POU3F2, which is important for hypothalamic development and function. Using morpholino and mutant zebrafish models, we show that POU3F2 lies downstream of SIM1 and controls oxytocin expression in the hypothalamic neuroendocrine preoptic area. We show that this finding is consistent with the expression patterns of POU3F2 and related genes in the human brain. Our work helps to further delineate the neuro-endocrine control of energy balance/body mass and demonstrates that this molecular pathway is conserved across multiple species.

Pharmacological treatment and BBB-targeted genetic therapy for MCT8-dependent hypomyelination in zebrafish.

ABSTRACTHypomyelination is a key symptom of Allan-Herndon-Dudley syndrome (AHDS), a psychomotor retardation associated with mutations in the thyroid-hormone (TH) transporter MCT8 (monocarboxylate transporter 8). AHDS is characterized by severe intellectual deficiency, neuromuscular impairment and brain hypothyroidism. In order to understand the mechanism for TH-dependent hypomyelination, we developed an mct8 mutant (mct8−/−) zebrafish model. The quantification of genetic markers for oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes revealed reduced differentiation of OPCs into oligodendrocytes in mct8−/− larvae and adults. Live imaging of single glial cells showed that the number of oligodendrocytes and the length of their extensions are reduced, and the number of peripheral Schwann cells is increased, in mct8−/− larvae compared with wild type. Pharmacological analysis showed that TH analogs and clemastine partially rescued the hypomyelination in the CNS of mct8−/− larvae. Intriguingly, triiodothyronine (T3) treatment rescued hypomyelination in mct8−/− embryos before the maturation of the blood–brain barrier (BBB), but did not affect hypomyelination in older larvae. Thus, we expressed Mct8-tagRFP in the endothelial cells of the vascular system and showed that even relatively weak mosaic expression completely rescued hypomyelination in mct8−/− larvae. These results suggest potential pharmacological treatments and BBB-targeted gene therapy that can enhance myelination in AHDS and possibly in other TH-dependent brain disorders.

Ptk7 mutant zebrafish models of congenital and idiopathic scoliosis implicate dysregulated Wnt signalling in disease

Scoliosis is a complex genetic disorder of the musculoskeletal system, characterized by three-dimensional rotation of the spine. Curvatures caused by malformed vertebrae (congenital scoliosis (CS)) are apparent at birth. Spinal curvatures with no underlying vertebral abnormality (idiopathic scoliosis (IS)) most commonly manifest during adolescence. The genetic and biological mechanisms responsible for IS remain poorly understood due largely to limited experimental models. Here we describe zygotic ptk7 (Zptk7) mutant zebrafish, deficient in a critical regulator of Wnt signalling, as the first genetically defined developmental model of IS. We identify a novel sequence variant within a single IS patient that disrupts PTK7 function, consistent with a role for dysregulated Wnt activity in disease pathogenesis. Furthermore, we demonstrate that embryonic loss-of-gene function in maternal-zygotic ptk7 mutants (MZptk7) leads to vertebral anomalies associated with CS. Our data suggest novel molecular origins of, and genetic links between, congenital and idiopathic forms of disease.