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Updated: Feb 17, 2026
Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish
Published on: October 27, 2017
Transcriptomic profiling of mTOR and ryanodine receptor signaling molecules in developing zebrafish in the absence
Daniel F Frank1,2, Galen W Miller3, Richard E Connon1
1Department of Anatomy, Physiology and Cell Biology, University of California, Davis, CA, USA.
Abstract:
The mechanistic target of rapamycin (mTOR) and ryanodine receptor (RyR) signaling pathways regulate fundamental processes of neurodevelopment, and genetic mutations within these pathways have been linked to neurodevelopmental disorders. While previous studies have established that these signaling molecules are expressed in developing zebrafish, a detailed characterization of the ontogenetic profile of these signaling molecules is lacking. Thus, we evaluated the spatiotemporal expression of key transcripts in mTOR and RyR signaling pathways in wildtype zebrafish at 24, 72 and 120 hours post fertilization (hpf). We further determined whether transcriptional profiles of a subset of genes in both pathways were altered by exposure to PCB 95 (2,2',3,5',6-pentachlorobiphenyl), a pervasive environmental contaminant known to cause developmental neurotoxicity in mammalian systems via RyR-dependent mechanisms. Quantitative PCR revealed that transcription generally increased across development. Genes in the signaling pathway upstream of the mTORC1 complex, and the RyR-paralogs, ryr2a and ryr3, were robustly upregulated, and in situ hybridization of ryr3 coincided with a transcriptional shift from muscle to neuronal tissue after 24 hpf. Static waterborne exposure to PCB 95 beginning at 6 hpf significantly altered transcription of genes in both pathways. These changes were concentration- and time-dependent, and included downregulation of rptor, a member of the mTORC1 complex, at both 72 and 120 hpf, and increased transcript levels of the RyR paralog ryr2b and downstream target of RyR signaling, Wingless-type 2ba (wnt2ba) at 72 hpf. The detailed transcriptomic profiling of key genes within these two signaling pathways provides a baseline for identifying other environmental factors that modify normal spatiotemporal expression patterns of mTOR and RyR signaling pathways in the developing zebrafish, as illustrated here for PCB 95.
Insights
This study maps the expression of mTOR and RyR pathway genes during zebrafish development and reveals how PCB 95 exposure alters these crucial neurodevelopmental pathways.
Area of Science:
- Neuroscience
- Developmental Biology
- Toxicology
Background:
- The mechanistic target of rapamycin (mTOR) and ryanodine receptor (RyR) signaling pathways are critical for neurodevelopment.
- Mutations in these pathways are associated with neurodevelopmental disorders.
- Zebrafish are a valuable model for studying developmental neurotoxicity.
Purpose of the Study:
- To characterize the spatiotemporal expression of key mTOR and RyR pathway transcripts during zebrafish development.
- To investigate the impact of PCB 95 exposure on these pathways in developing zebrafish.
Main Methods:
- Quantitative PCR (qPCR) was used to analyze gene expression at 24, 72, and 120 hours post fertilization (hpf).
- In situ hybridization was employed to visualize the spatial expression of specific genes.
- Zebrafish embryos were exposed to varying concentrations of PCB 95 starting at 6 hpf.
Main Results:
- Gene transcription generally increased throughout zebrafish development.
- Upregulation of mTOR pathway upstream genes and RyR paralogs (ryr2a, ryr3) was observed.
- PCB 95 exposure altered gene transcription in both pathways in a concentration- and time-dependent manner, including downregulation of rptor and upregulation of ryr2b and wnt2ba.
Conclusions:
- This study provides a comprehensive transcriptomic profile of mTOR and RyR signaling during zebrafish development.
- PCB 95 exposure disrupts key neurodevelopmental pathways in zebrafish.
- This work establishes a foundation for identifying environmental factors affecting neurodevelopmental gene expression.
