The Knockdown of 12-lipoxygenase in
Embryonic Zebrafish Causes Abnormal
Development
Amber Bannon
Mentor: Maret Traber, PhD
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
12 hpf
48 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 fluorescein tag to verify incorporation
 Knock down protein expression by blocking exon/intron
splice site
In normal cells…
EXON 1
INTRON
EXON 1
EXON 2
PROTEIN
EXON 2
Morpholino injected…
EXON 1
MORPHOLINO
INTRON
EXON 2
Control
EXON 1
MORPHOLINO
INTRON
Confirmation of block by RTPCR/gel electrophoresis
Injected
EXON 2
171bp
ABNORMAL
PROTEIN
250bp
LOX knockdown malformations
LOX morpholino
injected
Control
morpholino
injected
Noninjected
control
LOX morpholino injection increases the
number of embryonic malformations
Trial 1
16
14
12
10
8
Malformed
Dead
6
4
2
48hpf
72hpf
120hpf
Noninj
Control
LOX
Noninj
Control
LOX
Noninj
Control
0
LOX
Number of fish
18
LOX morpholino injection increases the
number of embryonic malformations
Trial 2
35
30
25
20
15
Malformed
Dead
10
5
48hpf
96hpf
120hpf
Noninj
Control
LOX
Noninj
Control
LOX
Noninj
Control
0
LOX
Number of fish
40
LOX morpholino injection increases the
number of embryonic malformations
Trial 3
16
14
12
10
8
Malformed
Dead
6
4
2
48hpf
72hpf
96hpf
Noninj
Control
LOX
Noninj
Control
LOX
Noninj
Control
LOX
Noninj
Control
0
LOX
Number of fish
18
120hpf
Quantitative Reverse TranscriptasePolymerase 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
Proposed pathway for lipoxygenase
PL-Arachidonic
activity
LOX
PL-HPETE
GPx4
PLA2
PL-HETE
HPETE
PLA2
GPx4
HETE
12-lipoxygenase (12-LOX)
 Catalyzes the addition of oxygen to the 12th carbon of
arachidonic acid
 Produces hydroperoxides (HpETE) in phospholipids
 Important role in normal biological functions
 Byproducts cause the lipid-oxidation chain reaction
OOH
Arachidonic acid
12-HpETE
12-lipoxygenase
Proposed pathway for lipoxygenase
PL-Arachidonic
activity
LOX
PL-HPETE
GPx4
PLA2
PL-HETE
HPETE
PLA2
GPx4
HETE
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
Proposed pathway for lipoxygenase
PL-Arachidonic
activity
LOX
PL-HPETE
GPx4
PLA2
PL-HETE
HPETE
PLA2
GPx4
HETE
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