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Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants
Published on: June 1, 2009
Mimicking TGFBI Hot-Spot Mutation Did Not Result in Any Deposit Formation in the Zebrafish Cornea
Fulya Yaylacıoğlu Tuncay1,2, Beril Talim3, Pervin Rukiye Dinçer2
1Medical Biology, Gülhane Medical Faculty, University of Health Sciences, Ankara, Turkey.
Purpose:
Mutations in transforming growth factor beta-induced (TGFBI) protein are associated with a group of corneal dystrophies (CDs), classified as TGFBI-associated CDs, characterized by deposits in the cornea. Mouse models were not proper in several aspects for modelling human disease. The goal of this study was to generate zebrafish mutants to investigate the corneal phenotype and to decide whether zebrafish could be a potential model for TGFBI-associated CDs.
Methods:
The conserved arginine residue, codon 117, in zebrafish tgfbi gene was targeted with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 method. Cas9 VQR variant was used with two target-specific sgRNAs to generate mutations. The presence of mutations was evaluated by T7 Endonuclease Enzyme (T7EI) assay and the type of the mutations were evaluated by Sanger sequencing. The mutant zebrafish at 3 months and 1 year of age were investigated under the microscope for corneal opacity and eye sections were evaluated histopathologically with hematoxylin-eosin, masson-trichrome and congo red stains for corneal deposits.
Results:
We achieved indel variation at the target sequence that resulted in p.Ser115_Arg117delinsLeu (c. 347_353delinsT) by nonhomology mediated repair in F1. This zebrafish mutation had the potential to mimic two disease-causing mutations reported in human cases previously: R124L and R124L + del125-126. Mutant zebrafish did not show any corneal opacity or corneal deposits at 3 months and 1 year of age.
Conclusion:
This study generated the first zebrafish model mimicking the R124 hot spot mutation in TGFBI-associated CDs. However, evaluations even at 1 year of age did not reveal any deposits in the cornea histopathologically. This study increased the cautions for modelling TGFBI-associated CDs in zebrafish in addition to differences in the corneal structure between zebrafish and humans.
Insights
Zebrafish models mimicking TGFBI-associated corneal dystrophies were created but showed no corneal deposits. This highlights challenges in using zebrafish for modeling these specific human corneal conditions.
Area of Science:
- Ophthalmology
- Genetics
- Developmental Biology
Background:
- Transforming growth factor beta-induced (TGFBI) protein mutations cause corneal dystrophies (CDs) with corneal deposits.
- Existing mouse models inadequately represent human TGFBI-associated CDs.
- Zebrafish offer a potential alternative model for studying these conditions.
Purpose of the Study:
- To generate zebrafish mutants for TGFBI-associated CDs.
- To investigate the corneal phenotype in these zebrafish models.
- To assess the suitability of zebrafish for modeling human TGFBI-associated CDs.
Main Methods:
- CRISPR/Cas9 gene editing targeted the conserved arginine residue (codon 117) in zebrafish tgfbi.
- Mutations were confirmed via T7EI assay and Sanger sequencing.
- Mutant zebrafish were histopathologically examined for corneal opacity and deposits at 3 months and 1 year of age.
Main Results:
- A zebrafish mutation (p.Ser115_Arg117delinsLeu) mimicking human R124 mutations was generated.
- No corneal opacity or deposits were observed in mutant zebrafish at either 3 months or 1 year.
- Histopathological analysis confirmed the absence of corneal deposits.
Conclusions:
- The study established the first zebrafish model for the TGFBI-associated CDs R124 hotspot mutation.
- Despite mimicking the mutation, the model did not develop observable corneal deposits, even at one year.
- Differences in corneal structure and disease mechanisms necessitate caution when modeling TGFBI-associated CDs in zebrafish.
