Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

McGurk, Patrick D.; Lovely, C. Ben; Eberhart, Johann K.

doi:10.3791/51190-v

Cited by 3 publications

(2 citation statements)

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“…Previous time-lapse imaging studies typically utilize confocal laser scanning microscopy. 29,30,31,32 Here, we present the use of a multi-photon technique, which has many advantages over traditional confocal microscopy. The basis of multi-photon microscopy is that two photons of longer infrared wavelengths are used to excite the fluorophore.…”

Section: Discussionmentioning

confidence: 99%

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Williams

Bohnsack

2017

JoVE

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Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

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Section: Discussionmentioning

confidence: 99%

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Williams

Bohnsack

2017

JoVE

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“…The purpose of this method is threefold. First, generating zebrafish mutant lines which consistently produce severe phenotypes enables downstream developmental studies like time-lapse recording 5 and transplantation 6…”

Section: Introductionmentioning

confidence: 99%

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Brooks¹,

Nichols²

2017

JoVE

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Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

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