title: Gene-specific differential response to anti-apoptotic therapies in zebrafish models of ocular coloboma.
abstract: OBJECTIVE
  We recently demonstrated that molecular therapy using aminoglycosides can overcome the underlying genetic defect in two zebrafish models of ocular coloboma and showed abnormal cell death to be a key feature associated with the optic fissure closure defects. In further studies to identify molecular therapies for this common congenital malformation, we now examine the effects of anti-apoptotic compounds in zebrafish models of ocular coloboma in vivo.
  
  
  METHODS
  Two ocular coloboma zebrafish lines (pax2.1/noi(tu29a) and lamb1/gup(m189)) were exposed to diferuloylmethane (curcumin) or benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD-fmk; a pan-caspase inhibitor) for up to 8 days post-fertilization. The effects of these compounds were assessed by morphology, histology, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and western blot analysis.
  
  
  RESULTS
  The size of the coloboma in gup zebrafish mutants treated with diferuloylmethane was greatly reduced. In treated mutants a reduction in TUNEL staining and a 67% decrease in activated caspase-3 protein were observed. The release of cytochrome c from the mitochondria into the cytosol was reduced fourfold by in vivo diferuloylmethane treatment, suggesting that the drug was acting to inhibit the intrinsic apoptotic pathway. Inhibition of caspases directly with zVAD-fmk also resulted in a similar reduction in coloboma phenotype. Treatment with either diferuloylmethane or zVAD-fmk resulted in a statistically significant 1.4 fold increase in length of survival of these mutant zebrafish (p<0.001), which normally succumb to the lethal genetic mutation. In contrast, the coloboma phenotype in noi zebrafish mutants did not respond to either diferuloylmethane or zVAD-fmk exposure, even though inhibition of apoptotic cell death was observed by a reduction in TUNEL staining.
  
  
  CONCLUSIONS
  The differential sensitivity to anti-apoptotic agents in lamb1-deficient and pax2.1-deficient zebrafish models, suggests that apoptotic cell death is not a final common pathway in all ocular coloboma genotypes. When considering anti-cell death therapies for ocular colobomatous defects attention should be paid to the genotype under investigation.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver BC, Canada. cge30@interchange.ubc.ca
authors.forename: Cheryl Y
authors.initials: CY
authors.lastname: Gregory-Evans
authors.forename: Mariya
authors.initials: M
authors.lastname: Moosajee
authors.forename: Xianghong
authors.initials: X
authors.lastname: Shan
authors.forename: Kevin
authors.initials: K
authors.lastname: Gregory-Evans
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000235:genetics*; D003103:Coloboma / Q000196:embryology / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D003474:Curcumin / Q000494:pharmacology*; D045304:Cytochromes c / Q000032:analysis; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004625:Embryo, Nonmammalian; D005123:Eye / Q000196:embryology / Q000378:metabolism* / Q000473:pathology; D014644:Genetic Variation; D020287:In Situ Nick-End Labeling; D008136:Longevity / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D029961:Zebrafish Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D045304:Cytochromes c; D053148:Caspase 3; D003474:Curcumin