Gene chip applications in environmental toxicology. · Gene chip applications in environmental...

Patrick Larkin Ph.D. Vice President-Research and Development

EcoArray Inc, Alachua, FL USA (386) 418-1400

[email protected]

Gene chip applications in environmental toxicology.

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Advantages of Gene chips

-Biomarkers of exposure

-Compound discrimination and quantification

-Bioavailability

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The company- what we do.

EcoArray Inc. is a company that manufactures gene chips and provides support and services

related to these products.

Our products and services are specifically tailored to the toxicology field.

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Existing technologies that can measure compounds

• Whole animal bioassays.

Technology

• In vitro assays (ie. YES assay).

• Water/sediment chemistry tests

Limitations • Fail to report on what is happening in an animal.

• Can not report on metabolites of compounds.

• Insensitive endpoints.

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Existing technologies that can measure compounds

• Whole animal bioassays.

Technology

• In vitro assays (ie. YES assay).

• Water/sediment chemistry tests

Limitations • Fail to report on what is happening in an animal.

• Can not report on metabolites of compounds.

• Insensitive endpoints.

… gene chips overcome these limitations.

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Small glass slides or membranes that contain

genetic material.

What are gene chips?

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How gene chips work- an overview

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How gene chips work- an overview

EcoArray programs

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How gene chips work -In more detail…

liver

cellDNA

mRNA (expressed genes)

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Compound present in the environmentOR

Very sensitive tests!!!

liver

cellDNA

mRNA (expressed genes)

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Measure mRNA using gene chips

•Thousands of genes can be spotted onto chips

• mRNA changes can be quantitated

• Changes in mRNA can be used for the early detection of compounds BEFORE adverse effects are observed in animals.

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Constant Exposure to 100 ng/L E2

Days of treatment 0 10 20 30 40 50

Vtg

mR

NA

(ng/

µg to

tal R

NA

)

0.01

0.1

1

10

100

1000

10000

Pla

sma

Vtg

(mg/

ml)

0.01

0.1

1

10

100

RNA Protein

Early detection

Denslow et al. 12

1212

Perc

ent M

easu

red

Effe

ct

Concentration x Time of Exposure

Popula

tion

Molecu

lar

(gene

)

100

50

0

Physio

logica

l

Irreversible effects

Reversible effects

Monitor for contaminants before irreversible effects occurs

Early detection

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Robotics

How gene chips are made

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Chip surface

mRNA

mRNA (expressed gene)

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How gene chips are used

Label mRNA

Extract tissue

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How gene chips are used

Obtain genetic fingerprint

Label mRNA

Extract tissue

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Model species

Fathead minnow freshwater species that is commonly used for toxicology testing.

Example: Biomarkers for exposure

Sheepshead minnow estuarine species that is commonly used for toxicology testing.

Example: Compound discrimination and quantification

Largemouth bass important game fish found throughout much of the United States.

Example: Bioavailability

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Experimental strategy

Controlled laboratory exposures

Genetic fingerprint database

Pollutant B Pollutant CPollutant A

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Experimental strategy

Controlled laboratory exposures

Genetic fingerprint database

Field site

Pollutant B Pollutant CPollutant A

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Fathead minnow

male

female

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FHMinnow chip

• 200 gene chip.

•Genes obtained from a variety of methods (cDNA libraries, directed cloning, and subtraction libraries).

• Genes are parts of multiple pathways.

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Un-exposed female

Un-exposed male

Male exposed to 400 ng/L of E2 for 48hrs

FHMChips®

Genetic biomarker for estrogens

Fathead minnow experiments

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Fathead minnow experiments

We are developing a 2000+ oligonucleotide based gene chip in fathead minnows with the EPA.

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Sheepshead minnow

male

female

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Strong estrogens

Weak estrogens

17-β estradiol Endosulfan

Control Ethinylestradiol Methoxychlor

NonylphenolDiethylstilbestrol

(65 ng/L)

(100 ng/L)

100 ng/L

(590 ng/L)

(5.6 ug/L)

11.8 ug/L

Compound discrimination

-SHMs exposed to several estrogenic compounds

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Compound discrimination

0.01

10000

1000

100

10

1

0. 1

Pixe

l uni

ts

(Fol

d in

duct

ion)

1.66 0.42

TEG Estradiol

Methoxychlor DES

EE2 Nonylphenol Endosulfan

TEGEstradiol

MethoxychlorDES

EE2NonylphenolEndosulfan

Low

mol

ecul

ar m

ass

prot

ein

2

Vtg

2

Cho

rioge

nin

3

Cho

rioge

nin

2

Vtg

1

ER-a

lpha

ND

96-C

Ubi

quiti

n-co

njug

atin

g en

zym

e 9

ND

107-

B

Unk

n. P

rot.,

(Acc

sn. #

AA

H10

857)

ND

1-E

ND

15-B

3

ND

98-E

Gly

cosy

late

redu

ctas

e

Nor

mal

izat

ion

gene

s

Hep

atic

lipa

se p

recu

rsor

HM

G-C

oA re

duct

ase

Alp

ha1-

mic

rogl

obul

in/b

ikun

in

prec

urso

r pro

tein

Bet

a ac

tin

Tran

sfer

rin

0.01

10000

1000

100

10

1

0. 1

Pixe

l uni

ts(F

old

indu

ctio

n)1.660.42

TEGEstradiol

MethoxychlorDES


TEGEstradiol

MethoxychlorDES


Low

mol

ecul

ar m

ass

prot

ein

2

Vtg

2

Cho

rioge

nin

3

Cho

rioge

nin

2

Vtg

1

ER-a

lpha

ND

96-C

Ubi

quiti

n-co

njug

atin

gen

zym

e 9

ND

107-

B

Unk

n. P

rot.,

(Acc

sn. #

AA

H10

857)

ND

1-E

ND

15-B

3

ND

98-E

Gly

cosy

late

redu

ctas

e

Nor

mal

izat

ion

gene

s

Hep

atic

lipa

se p

recu

rsor

HM

G-C

oA re

duct

ase

Alp

ha1-

mic

rogl

obul

in/b

ikun

inpr

ecur

sor p

rote

in

Bet

aac

tin

Tran

sfer

rin

Larkin et al, EHP 2003 27

2727

1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

l uni

ts

1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

l uni

ts

1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

l uni

ts

1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

l uni

ts

1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

l uni

ts

1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

l uni

ts

1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

l uni

ts

1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

l uni

ts

Vtg 2 Vtg 1

Choriogenin 3Choriogenin 2

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

ER-α Coagulation Factor XI

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

1 Concentration

(ng/L)

0 10 100 10001Concentration

(ng/L)

0 10 100 1000 1 Concentration

(ng/L)


(ng/L)

0 10 100 1000

1 Concentration

(ng/L)


(ng/L)

0 10 100 10001 Concentration

(ng/L)


(ng/L)

0 10 100 1000

1 Concentration

(ng/L)


(ng/L)

0 10 100 10001 Concentration

(ng/L)


(ng/L)


(ng/L)


(ng/L)

0 10 100 1000

Larkin et al, EHP 2003

Quantification

-SHMs exposed to several different doses of EE2

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Transferrin AMBP protein (α-1-microglobulin)

Beta actin

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

1 Concentration

(ng/L)


(ng/L)

0 10 100 1000

1 Concentration

(ng/L)


(ng/L)

0 10 100 1000

1 Concentration

(ng/L)


(ng/L)


(ng/L)


(ng/L)

0 10 100 1000

Larkin et al, EHP 2003

Quantification

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Largemouth bass model

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Area 7 field site

Eustis Muck Farm

OCPs levels in the muscles of fish :

Chlordane Dieldrin

p’p’-DDD p’p’-DDE p’p’-DDT

toxaphene

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LMBass gene chip

• 500 gene chip.

•Genes obtained from a variety of methods (cDNA libraries, directed cloning, differential display, and subtraction libraries).

• Genes are parts of multiple pathways.

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• 17-β estradiol exposure (2.5 mg/kg inject, 48 hrs).

• p’p-DDE (100 mg/kg inject, 48 hrs).

• 11-ketotestosterone (2 mg/kg inject, 48 hrs).

• Characterize reproductive cycle

• Site of investigation

`

Laboratory exposure

Largemouth bass model

Field analysis

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Estradiol Treated (2.5 mg/kg for 48 hrs)

Controls

Estradiol laboratory exposures

Larkin et al, CBP 2002 34

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Estradiol laboratory exposures

0.10

1.00

10.00

100.00

1000.00

EA-L

MB

-348

Hem

opex

in-li

ke p

rote

in

EA-L

MB

-122

EA-L

MB

-106

Alp

ha-2

-HS-

glyc

opro

tein

Prot

ein

disu

lfide

isom

eras

e Es

trog

en re

cept

or s

ubty

pe I

EA-L

MB

-423

Asp

artic

pro

teas

e

Vite

lloge

nin

3

Cho

rioge

nin

3

Cho

rioge

nin

2

Vite

lloge

nin

2A

Vite

lloge

nin

1 Fold

cha

nge

* * * **** * *** * **

P<0.05 P<0.01 * **


3535

DDE laboratory exposures

0.1

1

10

ER a

lpha

Prot

ein

disu

lfide

is

omer

ase

Vtg.

3

Asp

artic

pr

otea

se

Cho

rioge

nin

3

Cho

rioge

nin

2 Vtg.

2A

Vtg.

1

Pixe

l uni

ts

(Fol

d ch

ange

)


3636

11KT laboratory exposures

0.10

1.00

10.00

EA-L

MB

-348

Hem

opex

in-li

ke p

rote

in

EA-L

MB

-122

EA-L

MB

-106

Alp

ha-2

-HS-

glyc

opro

tein

Prot

ein

disu

lfide

isom

eras

e

Estr

ogen

rece

ptor

sub

type

I

EA-L

MB

-423

Asp

artic

pro

teas

e

Vite

lloge

nin

3

Cho

rioge

nin

3

Cho

rioge

nin

2

Vite

lloge

nin

2A

Vite

lloge

nin

1

Fold

cha

nge *

* * * *

P<0.05 P<0.01 * **

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Bass reproductive cycle

January April August

Need to define “normal” fingerprint pattern in bass before one can identify atypical gene expression patterns in the field

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Bass reproductive cycle

January April August

Study site

Need to define “normal” fingerprint pattern in bass before one can identify atypical gene expression patterns in the field

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4040

October February April

0

5

10

15

20

25


0

200

400

600

800

Months

Vtg 2.

Vtg 1.

Vtg 2A.

Vtg 3.

Aspartic protease

ZP3

ZP2

Months

Pixe

l uni

ts(F

old

chan

ge)

Bass reproductive cycle(females)

40

4141

Months

Pixe

l uni

ts(F

old

chan

ge)

Bass reproductive cycle(males)

No significant change in the vtgs and choriogenins.


0

5

10

15

20

25

41

Eustis (study site)

Deleon Springs (clean site)

Field analysis

No change in ZP’s No change in Vtg’s

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Field analysis

0.10

1.00

10.00

EA

-LM

B-0

28

EA

-LM

B-4

43

Ser

ine

prot

ease

inhi

bito

r 2b

EA

-LM

B-0

35

Per

leca

n (h

epar

an s

ulfa

te

prot

eogl

ycan

2)

EA

-LM

B-1

90

EA

-LM

B-3

99

AM

BP

pro

tein

pre

curs

or

EA

-LM

B-1

14

Tryp

sin

II pr

ecur

sor

Pixe

l uni

ts

* *

* ******* Study site

Clean

P<0.05*

43

4343

Gene ontology database

44

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Field analysis

0.10

1.00

10.00

EA

-LM

B-0

28

EA

-LM

B-4

43

Ser

ine

prot

ease

inhi

bito

r 2b

EA

-LM

B-0

35

Per

leca

n (h

epar

an s

ulfa

te

prot

eogl

ycan

2)

EA

-LM

B-1

90

EA

-LM

B-3

99

AM

BP

pro

tein

pre

curs

or

EA

-LM

B-1

14

Tryp

sin

II pr

ecur

sor

Pixe

l uni

ts

* *

* ******* Study site

Clean

P<0.05*

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Digestive pathways may be affected in

these animals

Field analysis

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Summary

-Gene chips can be used for biomarkers for exposure.

-Gene chips can be used to discriminate between compounds and can provide quantifiable data.

-Gene chips can provide information on bioavailability of compounds.

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University of Florida Nancy Denslow, Ph.D.

Kevin Kroll Tara Sabo, Ph.D.

Jamie Kelso Arianna Poston

Jaleh Khorsandian-Falleh

Bill Farmerie, Ph.D. Li Liu

Anuj Sahni

Funding: SBIR grant #1R43ESA011882-01 SBRP (P42 ES 07375)

EcoArray Inc Barbara Carter

Acknowledgements

Fish and Wildlife Conservation

Commision, Eustis FL Bill Johnson

Ira Adelman, PhD. Leroy Folmar Ph.D.

US EPA Michael Hemmer

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See www.ecoarray.com

Our website contains links to manuscripts using this technology and other information

Additional information

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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC

BiosensingBiosensing withwith ZebrafishZebrafish

ElwoodElwood LinneyLinney, Ph. D., Ph. D.

Molecular Genetics and MicrobiologyMolecular Genetics and Microbiology

Duke University Medical CenterDuke University Medical Center

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transgenesis imaging

gene discovery

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Assumptions we make:Assumptions we make:

1) toxicants are impacting upon normal, existing pathways

2) there can be a differential sensitivity to a toxicant depending upon whether the organism or target organ is developing or fully formed

3) there are common pathways in different organisms

4) differences between organisms should be represented by “differences” in their genomes

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Our changing view of “biosensors”Our changing view of “biosensors”

1) transgenic indicator mice for snapshots of “retinoic acid activity”

2) transgenic, fluorescent zebrafish for live 4-D studies of activity

3) using zebrafish themselves as indicators or sensors for toxicant events

4) using discovery microarray analysis for identifying genes affected

Use results to design newUse results to design new biosensorbiosensor transgenicstransgenics

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RARE -TKpr-β gal

RXR RAR

RA

Subset of retinoic acid receptor activity in reporter transgenic mouse:

Reporter transgenic miceReporter transgenic miceusing constructed retinoic acidusing constructed retinoic acidresponsive promoterresponsive promoter

Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 54

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relative size of embryos

Time-lapse 2 cell to 17 hours

mouse 9.5d to neonate

R.Kalstron and D. KaneSize relationships, developmental time, changingSize relationships, developmental time, changingin size with development:in size with development:

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Mouse andMouse and zebrafishzebrafish homeoboxhomeobox genes:genes:

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Black bars denote spinal nerves of brachial plexus

level of curved line represents the level of limb or fin

shaded somites represent level of Hoxc-6 expression


Parallels in axial development between vertebrate species:Parallels in axial development between vertebrate species:

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Vol. 10, Issue 12, 1890-1902, December 2000

Zebrafish Comparative Genomics and the Origins of Vertebrate Chromosomes John H. Postlethwait,1,3 Ian G. Woods,2 Phuong Ngo-Hazelett,1 Yi-Lin Yan,1 Peter D. Kelly,2 Felicia Chu,2 Hui Huang,2 Alicia Hill-Force,1 and William S. Talbot2

SyntenicSyntenic relationshipsrelationshipsbetween vertebrate genomes --genes inherited as linked clusters during speciation

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retinoic acid responsive day 8.5 mouse embryo expressing lacZ from RARE TKpr sequences

24 hr live zebrafish embryo expressing YFP from RARE zGT2 basal promoter sequence

sizes approximately to scale

Retinoic acid indicator embryosRetinoic acid indicator embryos

RARERARE-promoter-ββgalgal/GFP/GFP/YFPYFPRXR RAR

RARA

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vitamin A(all-trans retinol)

retinaldehyde

retinoic acid [ligand for transcription factors]

4-oxoretinoic acid

Vitamin AVitamin A--Retinoid Relationships:Retinoid Relationships:

retinretinooll dehydrogenasedehydrogenase

retinretinaall dehydrogenase(RALDHdehydrogenase(RALDH))

cytochromecytochrome p450RAI(cyp26)p450RAI(cyp26)

RA synthesis

RA metabolism

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Dual in situ hybridization for RALDH2(purple) and cyp26a1(redDual in situ hybridization for RALDH2(purple) and cyp26a1(red--orange):orange):

head

tail

RALDH2 in somites

[cyp26 in growing caudal end]

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-Raldh2

- RA

- Cyp26A1

Somites

Neural tube tail bud

18hpf Embryo

Model of some of the retinoid events occurring in the trunk neurModel of some of the retinoid events occurring in the trunk neural tube:al tube:

RA

RA

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RALDH inhibitorRALDH inhibitor[our work][our work]

NecklessNeckless mutant inmutant inRALDH2RALDH2

DEAB

untreated Begemann, Schilling, Rauch, Geisler and Ingham

normal DEAB treated

Either chemical inhibition of Raldh’s in zebrafish with DEAB or an isolated Raldh2 zebrafish mutant produced phenotypes paralleling the mouse Raldh2 knockout phenotypes:

hindbrain changes forelimb or fin inhibition

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InIn situssitus ofof zebrafishzebrafish Raldh2 at different developmental stages[KariRaldh2 at different developmental stages[Kari YacisinYacisin]:]:

tailbud stage ~10h 10 somites~14h

14 somites~16h 18 somites~18h

20 somites~<20h 23h

Point:Point:apparent turn-off of RALDH2 as neural tube ceases to grow

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cyp 26 knockout mice Sakai, Meno, Fujii, Nishino, Shiratori, Saijoh, Rossant, and Hamada

cyp26 knockout mice Abu-Abed, Dolle, Metzger Beckett, Chambon and Petkovich [somewhat similar phenotypes from these authors]

Two mouse KO studies ofTwo mouse KO studies ofcyp26A1 revealed caudal truncationscyp26A1 revealed caudal truncationsand occasionaland occasional exencephalyexencephaly

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Summary:Summary:

1)Raldh2 and cyp26a1(and cyp26b1) can be found adjacent to each other in the developing embryo creating functional "microgradients" of RA ligand for RAR activity

2)expression patterns and available mutants for these genes in mouse and zebrafish show consider homology

3)in zebrafish the Raldh2 promoter is directly repressed by RA and the cyp26a1 promoter is directly induced by RA

4)this system is being studied to determine whether there might be a genetic and/or environmental basis for neural tube defects

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8h 10h 11.3h 13h 15.5h

17h 19h 21.5h 24h

Developmental changes flanking and including neural tube growthDevelopmental changes flanking and including neural tube growthwhich we arewhich we are analysinganalysing with 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24hwith 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24hmicroarraymicroarray analysis:analysis:

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Progression ofProgression of microarraymicroarray development:development:

1) oligos from 500 selected zebrafish genes

2)16k oligomer library from Compugen arrayed

3)now examining 22k zebrafish oligomer array produced by Agilent, bioinformatics through Paradigm

Affymetrix has produced zebrafish arrays but we have yet to use them

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Genes regulated during segmentationGenes regulated during segmentation[work done in collaboration with R. Malek at TIGR]

expression log base 2

upregulated at 12 hpf

downregulated at 12hpf

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Some elements of ourSome elements of our zebrafishzebrafish toolbox:toolbox:

1) live, fluorescent, transgenic embryos

2) anti-sense morpholino knockdown of gene expression

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Some transgenic lines we made:Some transgenic lines we made:

GFP off constitutive promoter

GFP off artificial construct--lights up cells migrating along pronephric ducts

YFP of zHuC promoter that lights up developing nervous system

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Four transgenic lines for examining nervous system:Four transgenic lines for examining nervous system:

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AUG 5'

AAAAAAAAAAUAC

m orpho lino

A. Morpholino t o inhibit t ranslat ion

B. Morpholino t o inhibit splicing

exon a exon b exon c exon d

exon a exon b exon d exon a exon b exon c exon d

m orpho lino against sp lice accept or

norm al spliced m RNA

abnorm al sp liced product due t o m orpho lino

AntisenseAntisense morpholinomorpholino approaches use byapproaches use byzebrafishzebrafish researchers to “knockdown” genesresearchers to “knockdown” genes

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nono--tailtail (T(T--allele)allele) morpholinomorpholino we injected into a 1we injected into a 1--cellcellzebrafishzebrafish embryoembryo----these are 4 day larvae afterthese are 4 day larvae afterhatchinghatching----the phenotype is what is seen with realthe phenotype is what is seen with realmutants in nomutants in no--tailtail

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ChlorpyrifosChlorpyrifos studiesstudies

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Acetycholinesterase

from Soreq and Seidman, Nature Reviews Neuroscience(2001)

classical function of hydrolysing the neurotransmitter, acetylcholine

micemice have AChE plus a butyrylcholinesterase so mouse KOs in AChE allow animals to at least be born and live ~21 days

zebrafishzebrafish only has AChE, so AChE-/-embryos show defects in muscle fiber formation, innervation, and primary sensory neurons die prematurely--embryos are initially motile and then develop paralysis and die

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ChlorpyrifosChlorpyrifos treatingtreating zebrafishzebrafish embryosembryos----our workour work, high dose(500ng/ml):, high dose(500ng/ml):

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Experimental Plan:Experimental Plan:1) expose embryos for 5 days with

chlorpyrifos

2) adult learning studies in E. Levin’s lab

3) acetylcholine esterase assays during embryogenesis

4) AChE morpholino titration to CPF inhibition studies

5) adult learning studies and microarray analysis

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Averaged AChE Activity (0-5day exposure, 3 sets)

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9

Age at Extraction

AChE

Act

ivity

(a

rbitr

ary

units

) 0 ng/ml CPF 10 ng/ml CPF

100 ng/ml CPF

Acetylcholine esterase activity/Acetylcholine esterase activity/chlorpyrifoschlorpyrifos exposure:exposure:

79

7979


40

45

50

55

60

65

70

0 10 100

Chlorpyrifos (ng/ml)

Developmental Chlorpyrifos Exposure Effects on Average Choice Accuracy

* **

vs. Control (0 ng.ml CPF)* p<0.05** p<0.01

Collaborative work with E. Levin and E.Collaborative work with E. Levin and E. ChrysanthisChrysanthis::

As part of our Superfund program we chlorpyrifos treated embryos for 5 days, released them and grew them up and they tested for learning in maze designed by E. Levin in our Psychiatry department:

80

8080


Contol, MO, 100ng/ml CPF f.5/19/03

0 5

10 15 20 25 30 35 40 45

1 2 3 4 5 6 7 8 9

age at extraction

Rel

ativ

e A

ChE

Act

ivity

Control 0-5d DMSO MO-ache inj 100 ng/ml CPF 0-5d

Targeting acetylcholine esterase with aTargeting acetylcholine esterase with a morpholinomorpholino::

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8181


-20

-15

-10

-5

0

5

Percent Correct Difference from Controls

CPF 0 CPF 10 CPF 100 Morpholino Control Morpholino

Delayed Spatial Alternation Choice Accuracywith Developmental Chlorpyrifos or Morphoino Zebrafish

vs. CPF 0 * p<0.05** p<0.01

vs. Morpholino Control# p<0.025

#

*

**

[work done in collaboration with E. Levin laboratory]

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8282


H56788

BM

095875

AI8

78728

BI8

66260

BG

304327

BE016746

AA606013

AW

058804

BE201806

AI7

93723

BG

308438

BG

985503

AI8

78080

AI6

41757

BI6

73358

BI8

80088

BM

070938

AI3

31953

MO-ache

10ng/ml

0

5

10

15

20

25

30

MO-ache

100ng/ml

10ng/ml

Preliminary comparison of expression of MOPreliminary comparison of expression of MO--AChEAChE, 100ng/ml CPF, 100ng/ml CPF10ng/ml CPF versus control using filter of 2x or above expressio10ng/ml CPF versus control using filter of 2x or above expression:n:

expression of experimental divided by control

each bar individual gene (15 out of 37 genes overlap of MO vs. 100ng/ml CPF)

[3 day treated and untreated embryos]

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8383


our zCyp26A1pr transgenics with RA inducible promoter

FutureFuture----Biosensors:Biosensors:

line 1 with 1 uM RA (18h-22h)

1) the generation of a series of responsive transgenics to small molecules (in progress, estrogen inducibility)

2) the use of the Sanger Centre zebrafish DNA assembly to identify clones for genes which show distinct responsiveness to environmental toxicants so that transgenics can be derived from their regulatory sequences

3) the analysis of the 22k array data to formulate potential pathways that toxicants are impacting upon

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Lab individuals involved in our CPF work: Neural tube work: Sue Donerly Lucia Upchurch Betsy Dobbs-McAuliffe Stephen Huang Margaret Lai

past members Kari Yacisin Keenan O’Leary Qingshun Zhao

Collaborators: Ed Levin Elizabeth Chrysanthis Renae Malek(TIGR) Brad Smith(MRM) G.A. Johnson(MRM)

Research support from:Research support from:NIEHS Superfund and Toxicogenomics Consortium, NICHD

[email protected] http://glowfish.mc.duke.edu

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http://glowfish.mc.duke.edu

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