Amber BannonMentor: 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 diseasesAtherosclerosisCancerOsteoporosis Diabetes

Kuhn and O’Donnell. Progress in lipid research 2006

Role in embryonic development has not yet been studied

HypothesisWe 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 motherGenes are generally homologous to those

in humansRapid embryonic development

48 hpf12 hpf

*hpf = hours post fertilization

Study designTreat embryos at the single cell stage

Inject with LOX morpholinoInject 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 injectionsShort 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

EXON 1 INTRON EXON 2

EXON 1 EXON 2

PROTEIN

In normal cells…

Morpholino injected…EXON 1 INTRON EXON 2

MORPHOLINO

EXON 2EXON 1 INTRONMORPHOLINO

ABNORMAL PROTEIN

Confirmation of block by RT-PCR/gel electrophoresis

171bp

250bp

Control

Injected

LOX knockdown malformations

LOX morpholino

injected

Control morpholino

injected

Noninjected control

LOX morpholino injection increases the number of embryonic malformations

LOX

Co

ntr

ol

No

nin

j

LOX

Co

ntr

ol

No

nin

j

LOX

Co

ntr

ol

No

nin

j

48hpf 72hpf 120hpf

0

2

4

6

8

10

12

14

16

18

Trial 1

MalformedDead

Nu

mb

er

of

fish

LOX morpholino injection increases the number of embryonic malformations

LOX

Co

ntr

ol

No

nin

j

LOX

Co

ntr

ol

No

nin

j

LOX

Co

ntr

ol

No

nin

j

48hpf 96hpf 120hpf

0

5

10

15

20

25

30

35

40

Trial 2

MalformedDead

Nu

mb

er

of

fish

LOX morpholino injection increases the number of embryonic malformations

LOX

Co

ntr

ol

No

nin

j

LOX

Co

ntr

ol

No

nin

j

LOX

Co

ntr

ol

No

nin

j

LOX

Co

ntr

ol

No

nin

j

48hpf 72hpf 96hpf 120hpf

0

2

4

6

8

10

12

14

16

18

Trial 3

MalformedDead

Nu

mb

er

of

fish

Quantitative Reverse Transcriptase- Polymerase Chain Reaction (qRT-PCR)Utilizes reverse transcriptase to

synthesis cDNA from mRNAAmplifies and quantifies cDNA in real

timeThe number of copies of cDNA

measure the relative gene expressionUsing control and morpholino injected

samples we are able to determine changes in gene expression

HPETE

PL-Arachidonic

LOX

PL-HPETE

GPx4 PLA2

PLA2

PL-HETE

GPx4

HETE

Proposed pathway for lipoxygenase activity

12-lipoxygenase (12-LOX)Catalyzes the addition of oxygen to the 12th

carbon of arachidonic acid Produces hydroperoxides (HpETE) in

phospholipidsImportant role in normal biological functions

Byproducts cause the lipid-oxidation chain reaction

Arachidonic acid

OOH12-HpETE

12-lipoxygenase

HPETE

PL-Arachidonic

LOX

PL-HPETE

GPx4 PLA2

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 functionPhospholipid antioxidant that utilizes

glutathione and selenium

OOH12-HpETE

OH12-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

PLA2

PL-HETE

GPx4

HETE

Proposed pathway for lipoxygenase activity

Phospholipase A2

ConclusionKnock 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

AcknowledgementsMaret Traber, Ph.D.Traber labGalen MillerEd LabutRobert Tanguay, Ph.D.Tanguay LabKevin Ahern, Ph.D.Howard Hughes Medical InstituteCripps Scholarship Fund, College of Science