title: An ENU mutagenesis screen in zebrafish for visual system mutants identifies a novel splice-acceptor site mutation in patched2 that results in Colobomas.
abstract: OBJECTIVE
  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 postfertilization (dpf) for ocular abnormalities, and 5 dpf 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 used to clone and characterize a coloboma mutant.
  
  
  RESULTS
  A total of 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, uta(1), 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. ptch2(uta1) mutants possessed elevated Hedgehog (Hh) pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2(uta1) 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.
authors.affiliation: Section of Molecular Cell and Developmental Biology, Institute of Cell and Molecular Biology, The University of Texas at Austin, Texas 78712, USA.
authors.forename: Jiwoon
authors.initials: J
authors.lastname: Lee
authors.forename: Ben D
authors.initials: BD
authors.lastname: Cox
authors.forename: Christina M S
authors.initials: CM
authors.lastname: Daly
authors.forename: Chanjae
authors.initials: C
authors.lastname: Lee
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Nuckels
authors.forename: Rachel K
authors.initials: RK
authors.lastname: Tittle
authors.forename: Rosa A
authors.initials: RA
authors.lastname: Uribe
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Gross
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003103:Coloboma / Q000235:genetics*; D004625:Embryo, Nonmammalian; D005038:Ethylnitrosourea / Q000633:toxicity*; D005123:Eye / Q000196:embryology; D005260:Female; D005808:Genes, Recessive; D053823:Hedgehog Proteins / Q000235:genetics; D017403:In Situ Hybridization; D007908:Lens, Crystalline / Q000002:abnormalities* / Q000196:embryology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D022821:RNA Splice Sites / Q000235:genetics*; D012160:Retina / Q000002:abnormalities* / Q000196:embryology; D017422:Sequence Analysis, DNA; D014704:Veratrum Alkaloids / Q000494:pharmacology; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D000477:Alkylating Agents; D053823:Hedgehog Proteins; D008565:Membrane Proteins; D022821:RNA Splice Sites; D014704:Veratrum Alkaloids; D029961:Zebrafish Proteins; C491715:ptch1 protein, zebrafish; D005038:Ethylnitrosourea; C000541:cyclopamine