High-throughput zebrafish histology

Sabaliauskas, Nicole; Foutz, Christina A.; Mest, Jason R.; Budgeon, Lynn R.; Sidor, Adam T.; Gershenson, Joseph A.; Joshi, Sanjay; Cheng, Keith C.

doi:10.1016/j.ymeth.2006.03.001

Cited by 123 publications

(98 citation statements)

References 3 publications

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“…The fish were rinsed in phosphate-buffered saline and dehydrated in graded ethanol. Agar blocks were prepared using zebrafish metal molds (32), and prefixed zebrafish were arranged in the agar blocks. The agar blocks were sent to the Veterinary Diagnostic Lab, Oregon State University, Corvallis, OR, for paraffin embedding and sectioning.…”

Section: Methodsmentioning

confidence: 99%

Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

Chatterjee

Padmanarayana

Abdullah

et al. 2015

Molecular and Cellular Biology

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Sensory hair cells convert mechanical motion into chemical signals.Otoferlin, a six-C2 domain transmembrane protein linked to deafness in humans, is hypothesized to play a role in exocytosis at hair cell ribbon synapses. To date, however, otoferlin has been studied almost exclusively in mouse models, and no rescue experiments have been reported. Here we describe the phenotype associated with morpholino-induced otoferlin knockdown in zebrafish and report the results of rescue experiments conducted with full-length and truncated forms of otoferlin. We found that expression of otoferlin occurs early in development and is restricted to hair cells and the midbrain. Immunofluorescence microscopy revealed localization to both apical and basolateral regions of hair cells. Knockdown of otoferlin resulted in hearing and balance defects, as well as locomotion deficiencies. Further, otoferlin morphants had uninflated swim bladders. Rescue experiments conducted with mouse otoferlin restored hearing, balance, and inflation of the swim bladder. Remarkably, truncated forms of otoferlin retaining the C-terminal C2F domain also rescued the otoferlin knockdown phenotype, while the individual N-terminal C2A domain did not. We conclude that otoferlin plays an evolutionarily conserved role in vertebrate hearing and that truncated forms of otoferlin can rescue hearing and balance.H air cells couple mechanical motion to neurotransmitter release at synapses (1). In contrast to conventional neural synapses, hair cell synapses release neurotransmitter continuously and, in a graded manner (2), possess synaptic ribbons (2-4), and lack synaptophysin (5), complexin (6-9), Munc13 (10), and the calcium sensors synaptotagmin I and II (11). In place of synaptotagmin, it is believed that otoferlin may confer calcium sensitivity to evoke neurotransmitter release (12, 13). Otoferlin is a six-C2 domain transmembrane protein expressed in inner, outer, and vestibular hair cells, as well as restricted regions of the brain (13-16). In humans, missense mutations in otoferlin have been linked to hearing loss (17,18), and biochemical studies have determined that otoferlin binds calcium and lipids (12,19), as well as membrane trafficking proteins (12,(20)(21)(22)(23). Further, in vitro assays have demonstrated that otoferlin accelerates SNARE-mediated membrane fusion (12). Based upon this evidence, it is hypothesized that otoferlin functions as a calcium-sensitive regulator of neurotransmitter release in sensory hair cells.However, the results of several studies have raised questions related to otoferlin's function. For instance, despite otoferlin expression in vestibular hair cells, knockout mice show no balance defects (24, 25) despite reduced exocytosis in vestibular type I hair cells (24,26). This raises questions as to the importance of otoferlin in this system. Further, otoferlin did not rescue synchronous neurotransmitter release in synaptotagmin I knockout cultured neurons, indicating that otoferlin and synaptotagmin are not functionally redundant (2...

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Section: Methodsmentioning

confidence: 99%

Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

Chatterjee

Padmanarayana

Abdullah

et al. 2015

Molecular and Cellular Biology

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“…Subsequently, they were transferred into an agarose mold as described before (Sabaliauskas et al, 2006) and aligned in the same orientation. The agarose block containing the larvae was then processed in an embedding machine (Medite TPC 15Trio) in an ascending series of ethanol, followed by xylol and finally paraffin.…”

Section: Qrt-pcrmentioning

confidence: 99%

Thyroid disruption in zebrafish (Danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions

et al. 2016

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The vertebrate thyroid system is important for multiple developmental processes, including eye development. Thus, its environmentally induced disruption may impact important fitnessrelated parameters like visual capacities and behaviour. The present study investigated the relation between molecular effects of thyroid disruption and morphological and physiological changes of eye development in zebrafish (Danio rerio). Two test compounds representing different molecular modes of thyroid disruption were used: propylthiouracil (PTU), which is an enzyme-inhibitor of thyroid hormone synthesis, and tetrabromobisphenol A (TBBPA), which interacts with the thyroid hormone receptors. Both chemicals significantly altered transcript levels of thyroid system-related genes (TR, TR, TPO, TSH, DIO1, DIO2 and DIO3) in a compound-specific way. Despite these different molecular response patterns, both treatments resulted in similar pathological alterations of the eyes such as reduced size, RPE cell diameter and pigmentation, which were concentration-dependent. The morphological changes translated into impaired visual performance of the larvae: the optokinetic response was significantly and concentrationdependently decreased in both treatments, together with a significant increase of light preference of PTU-treated larvae. In addition, swimming activity was impacted. This study provides first evidence that different modes of molecular action of the thyroid disruptors can be associated with uniform apical responses. Furthermore, this study is the first to show that pathological eye development, as it can be induced by exposure to thyroid disruptors, indeed translates into impaired visual capacities of zebrafish early life stages. Abstract:The vertebrate thyroid system is important for multiple developmental processes, including eye development. Thus, its environmentally induced disruption may impact important fitnessrelated parameters like visual capacities and behaviour. The present study investigated the relation between molecular effects of thyroid disruption and morphological and physiological changes of eye development in zebrafish (Danio rerio). Two test compounds representing different molecular modes of thyroid disruption were used: propylthiouracil (PTU), which is an enzyme-inhibitor of thyroid hormone synthesis, and tetrabromobisphenol A (TBBPA), which interacts with the thyroid hormone receptors. Both chemicals significantly altered transcript levels of thyroid system-related genes (TR, TR, TPO, TSH, DIO1, DIO2 and DIO3) in a compound-specific way. Despite these different molecular response patterns, both treatments resulted in similar pathological alterations of the eyes such as reduced size, RPE cell diameter and pigmentation, which were concentration-dependent. The morphological changes translated into impaired visual performance of the larvae: the optokinetic response was significantly and concentration-dependently decreased in both treatments, together with a significant increase of light preference of PT...

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“…The issues of working with this unique shape have inspired many methods that attempt to remedy these problems and deliver consistent, high-quality image capture. [1][2][3][4] However, all of these approaches still require substantial training to achieve proficiency, are laborious, and thus are often not practical for high-throughput screening or other applications. Quality imaging can be the most critical bottleneck in using the fish for many scientific areas.…”

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confidence: 99%

SCORE Imaging: Specimen in a Corrected Optical Rotational Enclosure

et al. 2010

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Visual data collection is paramount for the majority of scientific research. The added transparency of the zebrafish (Danio rerio) allows for a greater detail of complex biological research that accompanies seemingly simple observational tools. We developed a visual data analysis and collection approach that takes advantage of the cylindrical nature of the zebrafish allowing for an efficient and effective method for image capture that we call Specimen in a Corrected Optical Rotational Enclosure imaging. To achieve a nondistorted image, zebrafish were placed in a fluorinated ethylene propylene tube with a surrounding optically corrected imaging solution (water). By similarly matching the refractive index of the housing (fluorinated ethylene propylene tubing) to that of the inner liquid and outer liquid (water), distortion was markedly reduced, producing a crisp imagable specimen that is able to be fully rotated 3608. A similar procedure was established for fixed zebrafish embryos using convenient, readily available borosilicate capillaries surrounded by 75% glycerol. The method described here could be applied to chemical genetic screening and other related high-throughput methods within the fish community and among other scientific fields.

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High-throughput zebrafish histology

Cited by 123 publications

References 3 publications

Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

Thyroid disruption in zebrafish (Danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions

SCORE Imaging: Specimen in a Corrected Optical Rotational Enclosure

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