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The study of neurogenesis is essential to understanding fundamental developmental processes and for the development of cell replacement therapies for central nervous system disorders. Here, we designed an in vivo drug screening protocol in developing zebrafish to find new molecules and signalling pathways regulating neurogenesis in the ventral spinal cord. This unbiased drug screen revealed that 4 cyclooxygenase (COX) inhibitors reduced the generation of serotonergic interneurons in the developing spinal cord. These results fitted very nicely with available single‐cell RNAseq data revealing that floor plate cells show differential expression of 1 of the 2 COX2 zebrafish genes (ptgs2a). Indeed, several selective COX2 inhibitors and two different morpholinos against ptgs2a reduced the number of serotonergic neurons in the ventral spinal cord and led to locomotor deficits. Single‐cell RNAseq data and different pharmacological manipulations further revealed that COX2‐floor plate‐derived prostaglandin D2 promotes neurogenesis in the developing spinal cord by promoting mitotic activity in progenitor cells. Rescue experiments using a phosphodiesterase‐4 inhibitor suggest that intracellular changes in cAMP levels underlie the effects of COX inhibitors on neurogenesis and locomotion. Our study provides compelling in vivo evidence showing that prostaglandin signalling promotes neurogenesis in the ventral spinal cord. [ABSTRACT FROM AUTHOR]

Niemann Pick disease type C (NPC) is an autosomal recessive neurodegenerative lysosomal disorder characterized by an accumulation of lipids in different organs. Clinical manifestations can start at any age and include hepatosplenomegaly, intellectual impairment, and cerebellar ataxia. NPC1 is the most common causal gene, with over 460 different mutations with heterogeneous pathological consequences. We generated a zebrafish NPC1 model by CRISPR/Cas9 carrying a homozygous mutation in exon 22, which encodes the end of the cysteine-rich luminal loop of the protein. This is the first zebrafish model with a mutation in this gene region, which is frequently involved in the human disease. We observed a high lethality in npc1 mutants, with all larvae dying before reaching the adult stage. Npc1 mutant larvae were smaller than wild type (wt) and their motor function was impaired. We observed vacuolar aggregations positive to cholesterol and sphingomyelin staining in the liver, intestine, renal tubules and cerebral gray matter of mutant larvae. RNAseq comparison between npc1 mutants and controls showed 284 differentially expressed genes, including genes with functions in neurodevelopment, lipid exchange and metabolism, muscle contraction, cytoskeleton, angiogenesis, and hematopoiesis. Lipidomic analysis revealed significant reduction of cholesteryl esters and increase of sphingomyelin in the mutants. Compared to previously available zebrafish models, our model seems to recapitulate better the early onset forms of the NPC disease. Thus, this new model of NPC will allow future research in the cellular and molecular causes/consequences of the disease and on the search for new treatments. [ABSTRACT FROM AUTHOR]

Gene therapy is a promising therapeutic approach that has experienced significant groth in recent decades, with gene nanomedicines reaching the clinics. However, it is still necessary to continue developing novel vectors able to carry, protect, and release the nucleic acids into the target cells, to respond to the widespread demand for new gene therapies to address current unmet clinical needs. We propose here the use of zebrafish embryos as an in vivo platform to evaluate the potential of newly developed nanosystems for gene therapy applications in cancer treatment. Zebrafish embryos have several advantages such as low maintenance costs, transparency, robustness, and a high homology with the human genome. In this work, a new type of putrescinesphingomyelin nanosystems (PSN), specifically designed for cancer gene therapy applications, was successfully characterized and demonstrated its potential for delivery of plasmid DNA (pDNA) and miRNA (miR). On one hand, we were able to validate a regulatory effect of the PSN/miR on gene expression after injection in embryos of 0 hpf. Additionally, experiments proved the potential of themodel to study the transport of the associated nucleic acids (pDNA and miR) upon incubation in zebrafish water. The biodistribution of PSN/pDNA and PSN/miR in vivo was also assessed after microinjection into the zebrafish vasculature, demonstrating that the nucleic acids remained associated with the PSN in an in vivo environment, and could successfully reach disseminated cancer cells in zebrafish xenografts. Altogether, these results demonstrate the potential of zebrafish as an in vivo model to evaluate nanotechnology-based gene therapies for cancer treatment, as well as the capacity of the developed versatile PSN formulation for gene therapy applications. [ABSTRACT FROM AUTHOR]

NOP56 belongs to a C/D box small nucleolar ribonucleoprotein complex that is in charge of cleavage and modification of precursor ribosomal RNAs and assembly of the 60S ribosomal subunit. An intronic expansion in NOP56 gene causes Spinocerebellar Ataxia type 36, a typical late-onset autosomal dominant ataxia. Although vertebrate animal models were created for the intronic expansion, none was studied for the loss of function of NOP56. We studied a zebrafish loss-of-function model of the nop56 gene which shows 70% homology with the human gene. We observed a severe neurodegenerative phenotype in nop56 mutants, characterized mainly by absence of cerebellum, reduced numbers of spinal cord neurons, high levels of apoptosis in the central nervous system (CNS) and impaired movement, resulting in death before 7 days post-fertilization. Gene expression of genes related to C/D box complex, balance and CNS development was impaired in nop56 mutants. In our study, we characterized the first NOP56 loss-of-function vertebrate model, which is important to further understand the role of NOP56 in CNS function and development. [ABSTRACT FROM AUTHOR]

Background: Zebrafish (Danio rerio) is a model organism for the study of human cancer. Compared with the murine model, the zebrafish model has several properties ideal for personalized therapies. The transparency of the zebrafish embryos and the development of the pigment-deficient "casper" zebrafish line give the capacity to directly observe cancer formation and progression in the living animal. Automatic quantification of cellular proliferation in vivo is critical to the development of personalized medicine. Methods: A new methodology was defined to automatically quantify the cancer cellular evolution. ZFTool was developed to establish a base threshold that eliminates the embryo autofluorescence, automatically measures the area and intensity of GFP (green-fluorescent protein) marked cells, and defines a proliferation index. Results: The proliferation index automatically computed on different targets demonstrates the efficiency of ZFTool to provide a good automatic quantification of cancer cell evolution and dissemination. Conclusion: Our results demonstrate that ZFTool is a reliable tool for the automatic quantification of the proliferation index as a measure of cancer mass evolution in zebrafish, eliminating the influence of its autofluorescence. [ABSTRACT FROM AUTHOR]

It is largely assumed that the teleost retina shows continuous and active proliferative and neurogenic activity throughout life. However, when delving into the teleost literature, one finds that assumptions about a highly active and continuous proliferation in the adult retina are based on studies in which proliferation was not quantified in a comparative way at the different life stages or was mainly studied in juveniles/young adults. Here, we performed a systematic and comparative study of the constitutive proliferative activity of the retina from early developing (2 days post-fertilisation) to aged (up to 3–4 years post-fertilisation) zebrafish. The mitotic activity and cell cycle progression were analysed by using immunofluorescence against pH3 and PCNA, respectively. We observed a decline in the cell proliferation in the retina with ageing despite the occurrence of a wave of secondary proliferation during sexual maturation. During this wave of secondary proliferation, the distribution of proliferating and mitotic cells changes from the inner to the outer nuclear layer in the central retina. Importantly, in aged zebrafish, there is a virtual disappearance of mitotic activity. Our results showing a decline in the proliferative activity of the zebrafish retina with ageing are of crucial importance since it is generally assumed that the fish retina has continuous proliferative activity throughout life. [ABSTRACT FROM AUTHOR]

Background: Cancer metastasis is a deathly process, and a better understanding of the different steps is needed. The shedding of circulating tumor cells (CTCs) and CTC-cluster from the primary tumor, its survival in circulation, and homing are key events of the metastasis cascade. In vitro models of CTCs and in vivo models of metastasis represent an excellent opportunity to delve into the behavior of metastatic cells, to gain understanding on how secondary tumors appear. Methods: Using the zebrafish embryo, in combination with the mouse and in vitro assays, as an in vivo model of the spatiotemporal development of metastases, we study the metastatic competency of breast cancer CTCs and CTC-clusters and the molecular mechanisms. Results: CTC-clusters disseminated at a lower frequency than single CTCs in the zebrafish and showed a reduced capacity to invade. A temporal follow-up of the behavior of disseminated CTCs showed a higher survival and proliferation capacity of CTC-clusters, supported by their increased resistance to fluid shear stress. These data were corroborated in mouse studies. In addition, a differential gene signature was observed, with CTC-clusters upregulating cell cycle and stemness related genes. Conclusions: The zebrafish embryo is a valuable model system to understand the biology of breast cancer CTCs and CTC-clusters. [ABSTRACT FROM AUTHOR]

New nanoparticles and biomaterials are increasingly being used in biomedical research for drug delivery, diagnostic applications, or vaccines, and they are also present in numerous commercial products, in the environment and workplaces. Thus, the evaluation of the safety and possible therapeutic application of these nanomaterials has become of foremost importance for the proper progress of nanotechnology. Due to economical and ethical issues, in vitro and in vivo methods are encouraged for the testing of new compounds and/or nanoparticles, however in vivo models are still needed. In this scenario, zebrafish (Danio rerio) has demonstrated potential for toxicological and pharmacological screenings. Zebrafish presents an innate immune system, from early developmental stages, with conserved macrophage phenotypes and functions with respect to humans. This fact, combined with the transparency of zebrafish, the availability of models with fluorescently labelled macrophages, as well as a broad variety of disease models offers great possibilities for the testing of new nanoparticles. Thus, with a particular focus on macrophage–nanoparticle interaction in vivo, here, we review the studies using zebrafish for toxicological and biodistribution testing of nanoparticles, and also the possibilities for their preclinical evaluation in various diseases, including cancer and autoimmune, neuroinflammatory, and infectious diseases. [ABSTRACT FROM AUTHOR]

The neural crest is a multipotent cell population that develops from the dorsal neural fold of vertebrate embryos in order to migrate extensively and differentiate into a variety of tissues. A number of gene regulatory networks coordinating neural crest cell specification and differentiation have been extensively studied to date. Although several publications suggest a common role for microRNA-145 (miR-145) in molecular reprogramming for cell cycle regulation and/or cellular differentiation, little is known about its role during in vivo cranial neural crest development. By modifying miR-145 levels in zebrafish embryos, abnormal craniofacial development and aberrant pigmentation phenotypes were detected. By whole-mount in situ hybridization, changes in expression patterns of col2a1a and Sry-related HMG box (Sox) transcription factors sox9a and sox9b were observed in overexpressed miR-145 embryos. In agreement, zebrafish sox9b expression was downregulated by miR-145 overexpression. In silico and in vivo analysis of the sox9b 3′UTR revealed a conserved potential miR-145 binding site likely involved in its post-transcriptional regulation. Based on these findings, we speculate that miR-145 participates in the gene regulatory network governing zebrafish chondrocyte differentiation by controlling sox9b expression. [ABSTRACT FROM AUTHOR]

Autosomal recessive ataxias are much less well studied than autosomal dominant ataxias and there are no clearly defined systems to classify them. Autosomal recessive ataxias, which are characterized by neuronal and multisystemic features, have significant overlapping symptoms with other complex multisystemic recessive disorders. The generation of animal models of neurodegenerative disorders increases our knowledge of their cellular and molecular mechanisms and helps in the search for new therapies. Among animal models, the zebrafish, which shares 70% of its genome with humans, offer the advantages of being small in size and demonstrating rapid development, making them optimal for high throughput drug and genetic screening. Furthermore, embryo and larval transparency allows to visualize cellular processes and central nervous system development in vivo. In this review, we discuss the contributions of zebrafish models to the study of autosomal recessive ataxias characteristic phenotypes, behavior, and gene function, in addition to commenting on possible treatments found in these models. Most of the zebrafish models generated to date recapitulate the main features of recessive ataxias. [ABSTRACT FROM AUTHOR]

Hereditary dominant ataxias are a heterogeneous group of neurodegenerative conditions causing cerebellar dysfunction and characterized by progressive motor incoordination. Despite many efforts put into the study of these diseases, there are no effective treatments yet. Zebrafish models are widely used to characterize neuronal disorders due to its conserved vertebrate genetics that easily support genetic edition and their optic transparency that allows observing the intact CNS and its connections. In addition, its small size and external fertilization help to develop high throughput assays of candidate drugs. Here, we discuss the contributions of zebrafish models to the study of dominant ataxias defining phenotypes, genetic function, behavior and possible treatments. In addition, we review the zebrafish models created for X-linked repeat expansion diseases X-fragile/fragile-X tremor ataxia. Most of the models reviewed here presented neuronal damage and locomotor deficits. However, there is a generalized lack of zebrafish adult heterozygous models and there are no knock-in zebrafish models available for these diseases. The models created for dominant ataxias helped to elucidate gene function and mechanisms that cause neuronal damage. In the future, the application of new genetic edition techniques would help to develop more accurate zebrafish models of dominant ataxias. [ABSTRACT FROM AUTHOR]

Published studies show that most of the human cancer xenograft studies in zebrafish embryos have used incubation temperatures in the range of 32–34 °C for 3–6 days post-injection, trying to find a compromise temperature between the zebrafish embryos (28 °C) and the human injected cells (37 °C). While this experimental setup is widely used, a question remains: is possible to overcome the drawbacks caused by a suboptimal temperature for the injected cells? To clarify the effect of temperature and injected cells on the host, in this study, we analyzed the development and health of the last in response to different temperatures in the presence or absence of injected human cancer cells. Comparing different incubation temperatures (28, 34 and 36 °C), we determined morphological abnormalities and developmental effects in injected and non-injected embryos at different time points. Besides this, the expression of selected genes was determined by qPCR to determine temperature affected metabolic processes in the embryos. The results indicate that an incubation temperature of 36 °C during a period of 48 h is suitable for xenotransplantation without morphological or metabolic changes that could be affecting the host or the injected cells, allowing them to proliferate near their optimal temperature. [ABSTRACT FROM AUTHOR]

Autism Spectrum Disorders (ASD) affect around 1.5% of the global population, which manifest alterations in communication and socialization, as well as repetitive behaviors or restricted interests. ASD is a complex disorder with known environmental and genetic contributors; however, ASD etiology is far from being clear. In the past decades, many efforts have been put into developing new models to study ASD, both in vitro and in vivo. These models have a lot of potential to help to validate some of the previously associated risk factors to the development of the disorder, and to test new potential therapies that help to alleviate ASD symptoms. The present review is focused on the recent advances towards the generation of models for the study of ASD, which would be a useful tool to decipher the bases of the disorder, as well as to conduct drug screenings that hopefully lead to the identification of useful compounds to help patients deal with the symptoms of ASD. [ABSTRACT FROM AUTHOR]

The first steps towards establishing xenografts in zebrafish embryos were performed by Lee et al., 2005 and Haldi et al., 2006, paving the way for studying human cancers using this animal species. Since then, the xenograft technique has been improved in different ways, ranging from optimizing the best temperature for xenografted embryo incubation, testing different sites for injection of human tumor cells, and even developing tools to study how the host interacts with the injected cells. Nonetheless, a standard protocol for performing xenografts has not been adopted across laboratories, and further research on the temperature, microenvironment of the tumor or the cell–host interactions inside of the embryo during xenografting is still needed. As a consequence, current non-uniform conditions could be affecting experimental results in terms of cell proliferation, invasion, or metastasis; or even overestimating the effects of some chemotherapeutic drugs on xenografted cells. In this review, we highlight and raise awareness regarding the different aspects of xenografting that need to be improved in order to mimic, in a more efficient way, the human tumor microenvironment, resulting in more robust and accurate in vivo results. [ABSTRACT FROM AUTHOR]

Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G > T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina. [ABSTRACT FROM AUTHOR]

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors. [ABSTRACT FROM AUTHOR]