Amber Bannon Mentor: Maret Traber, PhD The Knockdown of 12-lipoxygenase in Embryonic Zebrafish Causes Abnormal Development

Relevance 12-lipoxygenase may have a role in angiogenesis Lipoxygenase plays important roles in inflammatory diseases Atherosclerosis Cancer Osteoporosis Diabetes Kuhn and O’Donnell. Progress in lipid research 2006 Role in embryonic development has not yet been studied

Hypothesis We believe 12-lipoxygenase is necessary for normal embryonic development in zebrafish. Therefore, when it is knocked down, there will be an abnormal phenotype and altered gene expression.

Zebrafish model Able to separate embryo from mother Genes are generally homologous to those in humans Rapid embryonic development 48 hpf 12 hpf *hpf = hours post fertilization

Study design Treat embryos at the single cell stage Inject with LOX morpholino Inject with control morpholino (random oligomer) Non-injected control Observe individual embryos over a 5 day period Collect embryo samples for RNA extraction at 24 hpf and 48 hpf to analyze gene expression

Morpholino injections Short anti-sense oligomers with high mRNA binding affinity Labeled with f luorescein tag to verify incorporation Knock down protein expression by blocking exon/intron splice site

EXON 1 INTRON EXON 2 EXON 1EXON 2 PROTEIN In normal cells…

Morpholino injected… EXON 1 INTRON EXON 2 MORPHOLINO EXON 2EXON 1 INTRON MORPHOLINO ABNORMAL PROTEIN Confirmation of block by RT- PCR/gel electrophoresis 171bp 250bp ControlInjected

LOX knockdown malformations LOX morpholino injected Control morpholino injected Noninjected control

LOX morpholino injection increases the number of embryonic malformations

Quantitative Reverse Transcriptase- Polymerase Chain Reaction (qRT-PCR) Utilizes reverse transcriptase to synthesis cDNA from mRNA Amplifies and quantifies cDNA in real time The number of copies of cDNA measure the relative gene expression Using control and morpholino injected samples we are able to determine changes in gene expression

HPETE PL-Arachidonic LOX PL-HPETE GPx4 PLA2 PL-HETE GPx4 HETE Proposed pathway for lipoxygenase activity

12-lipoxygenase (12-LOX) Catalyzes the addition of oxygen to the 12 th carbon of arachidonic acid Produces hydroperoxides (HpETE) in phospholipids Important role in normal biological functions Byproducts cause the lipid-oxidation chain reaction Arachidonic acid OOH 12-HpETE

12-lipoxygenase

HPETE PL-Arachidonic LOX PL-HPETE GPx4 PLA2 PL-HETE GPx4 HETE Proposed pathway for lipoxygenase activity

Gluatathione peroxidase 4 (GPx4) Catalyzes the reduction of the hydroperoxides (HpETE) created by lipoxygenase Produces hydroxy products (HETE) Important signaling molecule for normal biological function Phospholipid antioxidant that utilizes glutathione and selenium OOH 12-HpETE OH 12-HETE

Glutathione Peroxidase 4

Phospholipase A2 (PLA2) Cleaves molecules from the phospholipid at the sn-2 position Produces HpETE and HETE from PL-HpETE and PL-HETE

HPETE PL-Arachidonic LOX PL-HPETE GPx4 PLA2 PL-HETE GPx4 HETE Proposed pathway for lipoxygenase activity

Phospholipase A2

Conclusion Knock down of 12-lipoxygenase expression causes an abnormal phenotype in zebrafish embryos There is a change in mRNA expression observed at 24 hpf This change appears to be linked to the abnormal morphology seen in lipoxygenase knock down embryos Lipoxygenase is necessary for normal embryogenesis in zebrafish

What’s next? This project provides an essential stepping stone for future research in the Traber lab on the molecular function of vitamin E in development Knock down the other genes in the proposed pathway Obtain vitamin E deficient fish and observe their development Knock down LOX in vitamin E deficient fish

Acknowledgements Maret Traber, Ph.D. Traber lab Galen Miller Ed Labut Robert Tanguay, Ph.D. Tanguay Lab Kevin Ahern, Ph.D. Howard Hughes Medical Institute Cripps Scholarship Fund, College of Science