A Zebrafish-Based Platform for High-Throughput Epilepsy Modeling and Drug Screening in F0
Abstract
:1. Introduction
2. Results
2.1. Tyrosinase Loss of Function Allows the Selection of Crispants Carrying High Rate of Mutations in Targeted Genes
2.2. adgrg1, gabra1, pcdh19, scn1lab and ube3a Crispants Show Epilepsy-like Behavior
2.3. Multiparametric Analysis of Behavioral Response to Light Flashes Allows Fine Characterization of Photosensitive Epilepsy
2.4. The Incubation of scn1lab Crispants with Antiepileptic Compounds Protects against Seizure-like Events
3. Discussion
3.1. An Optimized Workflow for Genetic Target Validation in Zebrafish F0 Larvae
3.2. Addressing the Phenotypic Heterogeneity of Epilepsy Combining Morphological and Behavioral Assays
3.3. The Characterization of Photosensitive Epilepsy with In-Depth Analysis of Larval Behavior
3.4. A High-Throughput Platform for ASM Drug Screening
3.5. Concluding Remarks
4. Materials and Methods
4.1. Zebrafish Husbandry and Breeding
4.2. Generation of Crispants
4.3. The Rate of Mutations Analysis
4.4. Morphological Analysis
- Plate A: scrambled, scn1lab and ube3a
- Plate B: scrambled, pcdh19 and gabra1
- Plate C: scrambled, kcnq2a, kcnq2b and kcnq2ab
- Plate D: scrambled and adgrg1
4.5. Behavioral Experiments
4.5.1. Locomotion Activity Assessment
- Plate A: scrambled, scn1lab and ube3a
- Plate B: scrambled, pcdh19 and gabra1
- Plate C: scrambled, kcnq2a, kcnq2b and kcnq2ab
- Plate D: scrambled and adgrg1
4.5.2. Light-Induced Epilepsy Behavior
- Plate A: scrambled, scn1lab and ube3a
- Plate B: scrambled, pcdh19 and gabra1
- Plate C: scrambled, kcnq2a, kcnq2b and kcnq2ab
- Plate D: scrambled and adgrg1
4.5.3. Pharmacological-Induced Epilepsy Behavior
- Plate A: scrambled, adgrg1, pcdh19 and ube3a
- Plate B: scrambled, scn1lab and gabra1
- Plate C: scrambled, kcnq2a, kcnq2b and kcnq2ab
4.6. Behavioral Analysis
4.7. Principal Component Analysis and Mahalanobis Distance Calculations
4.8. Pharmacology with Antiepileptic Compounds
- -
- Topiramate: 50 µM and 100 µM, treatment 180′ before the behavioral assay.
- -
- Valproic acid: 50 µM and 100 µM, treatment 180′ before the behavioral assay.
- -
- Fenfluramine: 17.5 µM and 35 µM, treatment 24 h before the behavioral assay.
4.9. The Statistics of the Pharmacology
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Scrambled | Tyrosinase | |||||
---|---|---|---|---|---|---|
Number of Positive | Total Number | % of Positive | Number of Positive | Total Number | % of Positive | |
Body curvature | 0 | 75 | 0 | 1 | 68 | 0.01 |
Snout jaw defects | 2 | 75 | 0.03 | 0 | 68 | 0 |
Yolk edema | 2 | 75 | 0.03 | 0 | 68 | 0 |
Necrosis | 1 | 75 | 0.01 | 2 | 68 | 0.03 |
Tail bending | 0 | 75 | 0 | 1 | 68 | 0.01 |
Notochord defects | 0 | 75 | 0 | 0 | 68 | 0 |
Craniofacial edema | 2 | 75 | 0.03 | 0 | 68 | 0 |
Fin absence | 0 | 75 | 0 | 0 | 68 | 0 |
Scoliosis | 0 | 75 | 0 | 0 | 68 | 0 |
Human Gene | Disease | Zebrafish Orthologue |
---|---|---|
ADGRG1 | Bilateral frontoparietal polymicrogyria [33] | adgrg1 |
GABRA1 | Different epileptic disorders [34] | gabra1 |
KCNQ2 | Benign familial neonatal seizures [35] | kcnq2a; kcnq2b |
PCDH19 | PCDH19 Epilepsy [36] | pcdh19 |
SCN1A | Dravet syndrome [37] | scn1lab |
UBE3A | Angelman syndrome [38] | ube3a |
Target Gene | Number of Larvae | Number of Flash Responses | Percentage of High-Activity Responses |
---|---|---|---|
scrambled | 116 | 617 | 5.1% |
adgrg1 | 86 | 282 | 8.9% |
gabra1 | 56 | 212 | 10.2% |
kcnq2a | 57 | 219 | 6% |
kcnq2b | 55 | 181 | 3% |
kcnq2a/kcnq2b | 53 | 208 | 0.6% |
pcdh19 | 50 | 157 | 8.5% |
scn1lab | 46 | 183 | 21.6% |
ube3a | 44 | 150 | 6.1% |
Zebrafish Gene | Primers | sgRNAs | Targeted Exon |
---|---|---|---|
adgrg1 | FW—5′-GTCATTTCTGTGTGTTCTGGGAG-3′ | 5′-CGGTGCAGCAGGTTCCTTGA-3′; | Exon 2 |
RV—5′-GGTGATGTTGTGATGCATGGTA-3′ | 5′-GTCAAAGGTGATATCATCAC-3′ | ||
gabra1 | FW—5′-TATTCCTTTGCACTGGCTGAGA-3′ | 5′-CTGCCTGAAGAACACATCTA-3′ | Exon 5 |
RV—5′-CGAACACAGACACCAACGAAAT-3′ | 5′-CCCGACACGTTCTTCCACAA-3′ | ||
kcnq2a | FW—5′-CCGCCAACGGGGAAGTTTA-3′ | 5′-GGTAAATGAACGCCCAGCCG-3′; | Exon 1 |
RV—5′-AGTTTGAGCATTCTGGGCGG-3′ | 5′-TCTGGAGCGACCCCGCGGCT-3′ | ||
kcnq2b | FW—5′-CCAGAACAAGTTCTCCAGGGA-3′ | 5′-TGCTCGCACCTGCTGTAGGG-3′; | Exon 1 |
RV—5′-AATTCTGCAGGCGTCGGTAA-3′ | 5′-TTTCTCGGCCTGCGGGGCGG-3′ | ||
pcdh19 | FW—5′-GGACTGGAGTCGATGCCG-3′ | 5′-TAACCCGCAAATAAGGCTGT-3′; | Exon 1 |
RV—5′-GTGTACCGAGACTGCGTTTCT-3′ | 5′-CGGTACCAGATTTCCCCTAG-3′ | ||
scn1lab | FW—5′-CATCAGCTCCCAGAGTGACC-3′ | 5′-TGCGATGCGTTGCTCGATAG-3′; | Exon 1 |
RV—5′-CCTTCAGGTAGTCCTACAGCCT-3′ | 5′-TCTCCGTAGATGAACGGCAG-3′ | ||
ube3a | FW—5′-ATCACAATGTGTACGCCGCT-3′ | 5′-GTTGTGGTCGCAGTACGGTG-3′; | Exon 2 |
RV—5′-GATCCACTCGAGGGCCTTTT-3′ | 5′-AGCTCGCTGGACTCAGGGAT-3′ |
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Locubiche, S.; Ordóñez, V.; Abad, E.; Scotto di Mase, M.; Di Donato, V.; De Santis, F. A Zebrafish-Based Platform for High-Throughput Epilepsy Modeling and Drug Screening in F0. Int. J. Mol. Sci. 2024, 25, 2991. https://doi.org/10.3390/ijms25052991
Locubiche S, Ordóñez V, Abad E, Scotto di Mase M, Di Donato V, De Santis F. A Zebrafish-Based Platform for High-Throughput Epilepsy Modeling and Drug Screening in F0. International Journal of Molecular Sciences. 2024; 25(5):2991. https://doi.org/10.3390/ijms25052991
Chicago/Turabian StyleLocubiche, Sílvia, Víctor Ordóñez, Elena Abad, Michele Scotto di Mase, Vincenzo Di Donato, and Flavia De Santis. 2024. "A Zebrafish-Based Platform for High-Throughput Epilepsy Modeling and Drug Screening in F0" International Journal of Molecular Sciences 25, no. 5: 2991. https://doi.org/10.3390/ijms25052991