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Journal: Zebrafish

22

Selection criteria for sperm cryopreservation are highly relevant in zebrafish since sperm quality is particularly variable in this species. Successful cryopreservation depends on high-quality sperm, which can only be ensured by the selection of breeders. Consequently, male selection and management are a priority to improve cryopreservation, and therefore, this study aimed to characterize optimal age and sperm collection frequency in zebrafish. For this purpose, males from wild type (AB) and from a transgenic line [Tg(runx2:eGFP)] were sampled at 6, 8, 12, and 14 months. For each age, sperm were collected at time 0 followed by samplings at 2, 7, and 14 days of rest. Sperm quality was assessed according to motility and membrane viability parameters. Quality assessment showed that Tg(runx2:eGFP) displayed significantly higher motility than AB and younger males showed higher motility in both lines. Sperm collection frequency affected membrane viability. While AB fish recovered sperm viability after 14 days of rest, Tg(runx2:eGFP) could not recover. Consequently, it may be important to study the sperm quality of each zebrafish line before sperm cryopreservation. Taking into consideration the results achieved in both lines, sperm collection should be performed between 6 and 8 months of age with a minimum collection interval of 14 days.

21

Zebrafish embryos and larvae have become popular vertebrate models because their body walls are transparent, which enables live imaging of target organs using fluorescent protein transgenes or dye staining. Software packages for the quantification of these fluorescent signals are available from both commercial and noncommercial sources; however, their algorithms are complicated and their resources (code) have mostly not been openly shared. In this study, we developed a simple and robust open-source software tool named “ZF-Mapper” for the quantification of the fluorescence intensity of each pixel in zebrafish images with batch image file processing capability. Using this software, we can evaluate the three-dimensional (3D) distribution of fluorescence intensity among zebrafish cells by analyzing each image pixel. We tested ZF-Mapper for the analysis of zebrafish with macrophage-specific enhanced green fluorescent protein (EGFP) and obtained results that were equivalent to those acquired using the conventional image analysis software ImageJ. We further applied ZF-Mapper to the analysis of zebrafish with cancer cell xenografts and quantified the amount of implanted melanoma cells labeled with a tdTomato red fluorescent protein in the whole body and the tail region. In addition, by combining ZF-Mapper with R freeware, we created an interactive 3D scatter plot of the fluorescence intensities of macrophage-EGFPs in zebrafish. In summary, we developed the Python-based freeware ZF-Mapper for the quantification of fluorescent signals in multiple zebrafish images, which enables fluorescence-based zebrafish screening. We provide the source code and the executable application software for Windows (.exe) and macOS (.app).

4

Glioblastoma multiforme is the most common and deadliest form of brain cancer. Glioblastomas are infiltrated by a high number of microglia, which promote tumor growth and surrounding tissue invasion. However, it is unclear how microglia and glioma cells physically interact and if there are differences, depending on glioma cell type. Hence, we have developed a novel live imaging assay to study microglia-glioma interactions in vivo in the zebrafish brain. We transplanted well-established human glioblastoma cell lines, U87 and U251, into transgenic zebrafish lines with labelled macrophages/microglia. Our confocal live imaging results show distinct interactions between microglia and U87, as well as U251 glioblastoma cells that differ in number and nature. Importantly these interactions do not appear to be antitumoral as zebrafish microglia do not engulf and phagocytose the human glioblastoma cells. Finally, xenotransplants into the irf8(-/-) zebrafish mutant that lacks microglia, as well as pharmacological inhibition of the CSF-1 receptor (CSF-1R) on microglia, confirm a prominent role for zebrafish microglia in promoting human glioblastoma cell growth. This new model will be an important tool for drug screening and the development of future immunotherapeutics targeting microglia within glioma.

Concepts: Protein, Cell, Cancer, Cell biology, Glioma, Brain tumor, Astrocytoma, Glioblastoma multiforme

3

Emotional disturbances constitute a major health issue affecting a considerable portion of the population in western countries. In this context, animal models offer a relevant tool to address the underlying biological determinants and to screen novel therapeutic strategies. While rodents have traditionally constituted the species of choice, zebrafish are now becoming a viable alternative. As zebrafish gain momentum in biomedical sciences, considerable efforts are being devoted to developing high-throughput behavioral tests. Here, we present a comparative study of zebrafish behavioral response to fear-evoking stimuli offered via three alternative methodologies. Specifically, in a binary-choice test, we exposed zebrafish to an allopatric predator Astronotus ocellatus, presented in the form of a live subject, a robotic replica, and a computer-animated image. The robot’s design and operation were inspired by the morphology and tail-beat motion of its live counterpart, thereby offering a consistent three-dimensional stimulus to focal fish. The computer-animated image was also designed after the live subject to replicate its appearance. We observed that differently from computer-animated images, both the live predator and its robotic replica elicited robust avoidance response in zebrafish. In addition, in response to the robot, zebrafish exhibited increased thrashing behavior, which is considered a valid indicator of fear. Finally, inter-individual response to a robotic stimulus is more consistent than that shown in response to live stimuli and animated images, thereby increasing experimental statistical power. Our study supports the view that robotic stimuli can constitute a promising experimental tool to elicit targeted behavioral responses in zebrafish.

Concepts: Psychology, Predation, Ecology, Experiment, Computer graphics, Robotics, Robot

2

Abstract Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.

Concepts: DNA, Cell nucleus, Cell, Enzyme, Embryo, Developmental biology, Stem cell, Cellular differentiation

1

Electronic databases provide effective and efficient management of zebrafish colony operations, but commercially available options are expensive. In this study we have developed a free zebrafish management repository alternative using free Google applications. Husbandry information is logged into a Google Sheets-based catalog through Google Form (GF) entries. Form autopopulation can be streamlined by barcodes, which can be generated and deciphered through free smartphone applications. The repository is capable of calculating pertinent husbandry dates from GF input and sending e-mail reminders to users for specified tasks. A Google application-based repository allows for a free simple zebrafish husbandry management solution.

0

The zebrafish has become an appropriate animal model in the analysis of numerous human brain disorders. A variety of neuropsychiatric conditions and neurodevelopmental disorders are comorbid with abnormal social behavior. Given the translational relevance of zebrafish, multidisciplinary studies employing behavioral, neurobiological, and molecular methods with this species may provide insights into human central nervous system (CNS) disorders. Many of these studies impinge upon our ability to properly induce and quantify the behavior of zebrafish, a relatively understudied aspect of this species. In this study, we investigate how the body size of conspecifics relative to that of the test subject influences social (shoaling) responses in zebrafish. We found a robust preference by wild-type (WT) test zebrafish toward big conspecifics, but not toward smaller conspecifics. Additionally, we tested an autism-relevant zebrafish knockout (KO) model. The dyrk1aa KO zebrafish showed impaired social preference compared with WT in the social behavior test. Our results confirm the effect of relative body size on social preference and that the social preference task developed for zebrafish may uncover the function of genes and biological mechanisms potentially associated with human CNS disorders.

0

In July 2018, the 11th Zebrafish Disease Models Conference (ZDM11) was held at Leiden University, The Netherlands, providing an excellent international opportunity for scientific presentations and collaborative discussion regarding the modeling of disease using zebrafish. Much like the original ZDM1, which was also hosted in Leiden in 2007, immunology and cancer had a strong presence at ZDM11, with zebrafish still proving an invaluable tool to interrogate their disease genetics and progression in vivo. In addition, ZDM11 built upon the inclusion and development of other key areas making use of zebrafish disease models, with sessions on neuroscience, behavior, muscle, skeletal and cardiac disease, and more. ZDM11 also highlighted the rapid progression and application of new and exciting technologies to assist in the generation and analysis of zebrafish disease models, including Crispr/Cas9 gene targeting tools, electroporation techniques, computational analysis, drug screening pipelines, and advances in vivo imaging such as high-resolution correlative electron microscopy and lightsheet microscopy. Here, we provide a summary of the ZDM11 conference proceedings, giving an overview of the stimulating science presented across 4 days and 13 conference sessions.

0

Electric fish of the order Gymnotiformes are endemic to the Neotropical region, and their highest diversity is observed in the Amazon region. The family Gymnotidae, which consists of the genera Electrophorus and Gymnotus, is a natural group and is located at the base of the phylogeny of the order. Gymnotus is a widely distributed and specious genus with high karyotypic diversity, especially concerning to the diploid number and the locations of repetitive sequences. Our karyotyping results in five species of the family Gymnotidae (Gymnotus ucamara, Gymnotus cf. stenoleucus, Gymnotus cf. pedanopterus, Gymnotus mamiraua, and Gymnotus carapo “Maranhão”) corroborate the proposal of plasticity of the diploid number in this group. Moreover, in this study, we propose that the 5S ribosomal DNA (rDNA) sequences were species-specific markers that act as a potential biogeographical marker for the genus. Besides, the sequence’s location, particularly in G. mamiraua from Central Amazon, shows a close relationship with 5S of the Gymnotus species, with 54 chromosomes, from the Paraná-Paraguay basin in the Center-South of Brazil. Considering that the ancestral diploid number for Gymnotidae is 52 chromosomes, we also suggest that the trend in the family is toward a decrease in the chromosome number. However, the carapo clade stands out in this regard, with an increase and a decrease in chromosome number; this pattern may be reinforced with the ecologic behaviors and the geodispersal patterns of this clade.

0

Fixation and decalcification can alter protein structure in tissues, influencing the efficacy of primary antibodies routinely used in immunohistochemical (IHC) staining. Histologic examination of zebrafish requires both processes, making staining and analysis potentially challenging. Here, we investigated the effects of common fixation and decalcification protocols on IHC staining in zebrafish. We also identified zebrafish-reactive and -specific antibodies for use in research and diagnostics. For several of the antibodies, time spent in Dietrich’s fixative containing 2% glacial acetic acid or 3.4% formaldehyde followed by decalcification with ethylenediaminetetraacetic acid (EDTA) significantly impacted IHC staining quality, particularly regarding staining intensity. Protocols utilizing shorter fixation times produced higher-quality stains. In addition, individual markers were variably affected by the type of fixative. Dietrich’s fixative significantly reduced staining quality for the “neural” markers: glial fibrillar acidic protein, chromogranin A, S100. A negative time-dependent effect of fixation on staining quality was found for several antibodies: muscle actin (Dietrich’s only), cytokeratin AE1/AE3, chromogranin, and S100. Neither decalcification protocol had a statistically significant negative time-dependent effect on staining quality. Based on our results, we suggest shorter fixation and decalcification protocols to best preserve IHC staining quality as well as recommend deliberate selection of the fixative used depending on the protein of interest.