Gene chip applications in environmental toxicology. · Gene chip applications in environmental...
Transcript of 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
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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How gene chips work -In more detail…
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
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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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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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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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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
EE2NonylphenolEndosulfan
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
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
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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)
0 10 100 10001Concentration
(ng/L)
0 10 100 1000
1 Concentration
(ng/L)
0 10 100 10001Concentration
(ng/L)
0 10 100 10001 Concentration
(ng/L)
0 10 100 10001Concentration
(ng/L)
0 10 100 1000
1 Concentration
(ng/L)
0 10 100 10001Concentration
(ng/L)
0 10 100 10001 Concentration
(ng/L)
0 10 100 10001Concentration
(ng/L)
0 10 100 10001Concentration
(ng/L)
0 10 100 10001Concentration
(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)
0 10 100 10001Concentration
(ng/L)
0 10 100 1000
1 Concentration
(ng/L)
0 10 100 10001Concentration
(ng/L)
0 10 100 1000
1 Concentration
(ng/L)
0 10 100 10001Concentration
(ng/L)
0 10 100 10001Concentration
(ng/L)
0 10 100 10001Concentration
(ng/L)
0 10 100 1000
Larkin et al, EHP 2003
Quantification
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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
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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 * **
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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
)
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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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October February April
0
5
10
15
20
25
October February April
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)
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Months
Pixe
l uni
ts(F
old
chan
ge)
Bass reproductive cycle(males)
No significant change in the vtgs and choriogenins.
October February April
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*
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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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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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
transgenesis imaging
gene discovery
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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
Parallels in axial development between vertebrate species:Parallels in axial development between vertebrate species:
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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
-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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
Four transgenic lines for examining nervous system:Four transgenic lines for examining nervous system:
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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
ChlorpyrifosChlorpyrifos studiesstudies
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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
ChlorpyrifosChlorpyrifos treatingtreating zebrafishzebrafish embryosembryos----our workour work, high dose(500ng/ml):, high dose(500ng/ml):
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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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:
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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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:
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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
-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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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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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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