An ENU Mutagenesis Screen in Zebrafish for Visual System Mutants Identifies a Novel Splice-Acceptor Site Mutation in patched2 that Results in Colobomas
- Authors
- Lee, J., Cox, B.D., Daly, C.M., Lee, C., Nuckels, R.J., Tittle, R.K., Uribe, R.A., and Gross, J.M.
- ID
- ZDB-PUB-121130-16
- Date
- 2012
- Source
- Investigative ophthalmology & visual science 53(13): 8214-8221 (Journal)
- Registered Authors
- Gross, Jeffrey, Lee, Chanjae, Lee, Jiwoon, Nuckels, Richard, Tittle, Rachel, Uribe, Rosa
- Keywords
- none
- MeSH Terms
-
- Alkylating Agents/toxicity
- Alleles
- Amino Acid Sequence
- Animals
- Base Sequence
- Coloboma/genetics*
- Embryo, Nonmammalian
- Ethylnitrosourea/toxicity*
- Eye/embryology
- Female
- Genes, Recessive
- Hedgehog Proteins/genetics
- In Situ Hybridization
- Lens, Crystalline/abnormalities*
- Lens, Crystalline/embryology
- Male
- Membrane Proteins/genetics*
- Molecular Sequence Data
- Mutagenesis/drug effects
- Mutation/genetics*
- Polymerase Chain Reaction
- RNA Splice Sites/genetics*
- Retina/abnormalities*
- Retina/embryology
- Sequence Analysis, DNA
- Veratrum Alkaloids/pharmacology
- Zebrafish
- Zebrafish Proteins/genetics*
- PubMed
- 23150614 Full text @ Invest. Ophthalmol. Vis. Sci.
Purpose: To identify recessive mutations affecting development and/or maintenance of the zebrafish visual system. Methods: A three-generation ENU (N-Nitroso-N-ethylurea)-based forward genetic screen was performed. F3 embryos were screened visually from 1 to 5 days-post-fertilization (dpf) for ocular abnormalities, and 5dpf embryos were fixed and processed for cryosectioning, after which eye sections were screened for defects in cellular organization within the retina, lens and cornea. A combination of PCR and DNA sequencing, in situ hybridization and pharmacological treatments were utilized to clone and characterize a coloboma mutant. Results: 126 F2 families were screened and from these, 18 recessive mutations were identified that affected eye development. Phenotypes included lens malformations and cataracts, photoreceptor defects, oculocutaneous albinism, microphthalmia and colobomas. Analysis of one such coloboma mutant, uta1, identified a splice-acceptor mutation in the patched2 gene that resulted in an in-frame deletion of 19 amino acids that are predicted to contribute to the first extracellular loop of Patched2. ptch2uta1 mutants possessed elevated Hh pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2uta1 mutant embryos. Conclusions: We have identified 18 recessive mutations affecting development of the zebrafish visual system and we have characterized a novel splice-acceptor site mutation in patched2 that results in enhanced Hh pathway activity and colobomas.