Nile Tilapia (Oreochromis niloticus) Patched1 Mutations Disrupt Cardiovascular Development and Vascular Integrity through Smoothened Signaling
Abstract
:1. Introduction
2. Results
2.1. cDNA Cloning and Sequence Analyses
2.2. Ptch1 Is Required for the Survival of Early Larvae of Nile Tilapia
2.3. ptch1 Mutations Cause Pericardial Edema and Abnormal Cardiac Morphology
2.4. ptch1 Mutations Impair Angiogenesis and Vascular Integrity
2.5. The Differential Expression Profiles of the ptch1 Mutant and WT Larvae
2.6. The Rescue of ptch1 Mutants by Smo Antagonist Treatment
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. Sample Collection, RNA Extraction, and cDNA Synthesis
4.3. Cloning and Sequence Analysis
4.4. Reverse Transcription PCR (RT-PCR) Analysis
4.5. In Situ Hybridization (ISH)
4.6. ptch1 Mutations by CRISPR/Cas9
4.7. Classification of Mutants and Calculation of Area
4.8. Hematoxylin and Eosin (H&E) Staining
4.9. RNA Sequencing (RNA-Seq)
4.10. Drug Treatment
4.11. Data Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Species | Full Length | Domain | ||||||
---|---|---|---|---|---|---|---|---|
Ncyto | Loop1 | SSD1 | MLcyto | Loop2 | SSD2 | Ccyto | ||
Oreochromis niloticus | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
Danio rerio | 76 | 86 | 81 | 99 | 55 | 85 | 97 | 53 |
Homo sapiens | 69 | 64 | 80 | 98 | 63 | 79 | 93 | 43 |
Group | Total Larvae Number | Number of Larvae with the Phenotype | Proportion of Larvae with High Mutation Rate (Phenotype of E+) | Proportion of Viable Larvae (Phenotype of C−) | ||
---|---|---|---|---|---|---|
C−, E− | C+, E+ | C−, E+ | ||||
WT | 336 | 336 | 0 | 0 | - | 100% |
10 μM Cyc | 239 | 239 | 0 | 0 | - | 100% |
ptch1 gRNA | 232 | 95 | 137 | 0 | 59.05% | 40.95% |
ptch1 gRNA +10 μM Cyc | 268 | 126 | 78 | 64 | 54.10% | 70.90% |
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Liu, X.; Zhao, C.; Liu, L.; Peng, X.; Li, J.; Tao, W.; Wang, D.; Wei, J. Nile Tilapia (Oreochromis niloticus) Patched1 Mutations Disrupt Cardiovascular Development and Vascular Integrity through Smoothened Signaling. Int. J. Mol. Sci. 2024, 25, 3321. https://doi.org/10.3390/ijms25063321
Liu X, Zhao C, Liu L, Peng X, Li J, Tao W, Wang D, Wei J. Nile Tilapia (Oreochromis niloticus) Patched1 Mutations Disrupt Cardiovascular Development and Vascular Integrity through Smoothened Signaling. International Journal of Molecular Sciences. 2024; 25(6):3321. https://doi.org/10.3390/ijms25063321
Chicago/Turabian StyleLiu, Xiang, Changle Zhao, Lei Liu, Xi Peng, Jianeng Li, Wenjing Tao, Deshou Wang, and Jing Wei. 2024. "Nile Tilapia (Oreochromis niloticus) Patched1 Mutations Disrupt Cardiovascular Development and Vascular Integrity through Smoothened Signaling" International Journal of Molecular Sciences 25, no. 6: 3321. https://doi.org/10.3390/ijms25063321