PUBLICATION
High-throughput detection of craniofacial defects in fluorescent zebrafish
- Authors
- Everson, J.L., Tseng, Y.C., Eberhart, J.K.
- ID
- ZDB-PUB-221115-55
- Date
- 2022
- Source
- Birth defects research 115(3): 371-389 (Journal)
- Registered Authors
- Eberhart, Johann
- Keywords
- birth defect, high-throughput screening, multifactorial interactions, neural crest, teratogen, toxicology, zebrafish
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- Hedgehog Proteins*/genetics
- Hedgehog Proteins*/metabolism
- Nitrosourea Compounds/metabolism
- Zebrafish*/genetics
- PubMed
- 36369674 Full text @ Birth Defects Res
Citation
Everson, J.L., Tseng, Y.C., Eberhart, J.K. (2022) High-throughput detection of craniofacial defects in fluorescent zebrafish. Birth defects research. 115(3):371-389.
Abstract
Losses and malformations of cranial neural crest cell (cNCC) derivatives are a hallmark of several common brain and face malformations. Nevertheless, the etiology of these cNCC defects remains unknown for many cases, suggesting a complex basis involving interactions between genetic and/or environmental factors. However, the sheer number of possible factors (thousands of genes and hundreds of thousands of toxicants) has hindered identification of specific interactions. Here, we develop a high-throughput analysis that will enable faster identification of multifactorial interactions in the genesis of craniofacial defects. Zebrafish embryos expressing a fluorescent marker of cNCCs (fli1:EGFP) were exposed to a pathway inhibitor standard or environmental toxicant, and resulting changes in fluorescence were measured in high-throughput using a fluorescent microplate reader to approximate cNCC losses. Embryos exposed to the environmental Hedgehog pathway inhibitor piperonyl butoxide (PBO), a Hedgehog pathway inhibitor standard, or alcohol (ethanol) exhibited reduced fli1:EGFP fluorescence at one day post fertilization, which corresponded with craniofacial defects at five days post fertilization. Combining PBO and alcohol in a co-exposure paradigm synergistically reduced fluorescence, demonstrating a multifactorial interaction. Using pathway reporter transgenics, we show that the plate reader assay is sensitive at detecting alterations in Hedgehog signaling, a critical regulator of craniofacial development. We go on to demonstrate that this technique readily detects defects in other important cell types, namely neurons. Together, these findings demonstrate this novel in vivo platform can predict developmental abnormalities and multifactorial interactions in high-throughput.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping