Gene chip applications in environmental toxicology.
Patrick Larkin Ph.D.Vice President-Research and Development
EcoArray Inc, Alachua, FL USA(386) 418-1400
1
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Advantages of Gene chips
-Biomarkers of exposure
-Compound discrimination and quantification
-Bioavailability
2
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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
Technology Limitations • Fail to report on what is • Water/sediment happening in an animal.chemistry tests
• In vitro assays • Can not report on
(ie. YES assay). metabolites of compounds.
• Whole animal bioassays. • Insensitive endpoints.
4
4 4
Existing technologies that can measure compounds
Technology Limitations • Fail to report on what is • Water/sediment happening in an animal.chemistry tests
• In vitro assays • Can not report on
(ie. YES assay). metabolites of compounds.
• Whole animal bioassays. • Insensitive endpoints.
… gene chips overcome these limitations.
5
5 5
How gene chips work -In more detail…
OR
liver
cellDNA
mRNA (expressed genes)
Compound present in the environment
Very sensitive tests!!! 10
10 10
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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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 A Pollutant B Pollutant C
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Experimental strategy
Controlled laboratory exposures
Field site
Genetic fingerprintdatabase
Pollutant A Pollutant B Pollutant C
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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.
22
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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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2323
Fathead minnow experiments
We are developing a 2000+ oligonucleotide based gene chip in fathead minnows with the EPA.
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2424
Compound discrimination Strong Weak
estrogens estrogens 17-β estradiol Endosulfan
-SHMs exposed to Control
several estrogenic compounds
Ethinylestradiol Methoxychlor
NonylphenolDiethylstilbestrol
(65 ng/L)
(100 ng/L)
100 ng/L
(590 ng/L)
(5.6 ug/L)
11.8 ug/L
26
26 26
Compound discrimination
1000010000
10001000
100100
Pixe
l uni
tsPi
xel u
nits
1010(F
old
indu
ctio
n)(F
old
indu
ctio
n)1.661.66
11 0.420.420.0. 11
0.010.01Tr
ansf
errin
Tr
ansf
errin
Bet
a ac
tin
Bet
aac
tin
Alp
ha1-
mic
rogl
obul
in/b
ikun
in
Alp
ha1-
mic
rogl
obul
in/b
ikun
inpr
ecur
sor p
rote
in
prec
urso
r pro
tein
HM
G-C
oA re
duct
ase
HM
G-C
oA re
duct
ase
Hep
atic
lipa
se p
recu
rsor
H
epat
ic li
pase
pre
curs
or
Nor
mal
izat
ion
gene
sN
orm
aliz
atio
n ge
nes
Low
mol
ecul
ar m
ass
prot
ein
2Lo
wm
olec
ular
mas
s pr
otei
n 2
TEGTEGTEGTEGEstradiol EE2Estradiol EE2Estradiol EE2Estradiol EE2
Gly
cosy
late
redu
ctas
eG
lyco
syla
te re
duct
ase
Methoxychlor NonylphenolMethoxychlor NonylphenolMethoxychlor NonylphenolMethoxychlor NonylphenolN
D98
-EN
D98
-EDES EndosulfanDES EndosulfanDES EndosulfanDES Endosulfan
ND
15-B
3N
D15
-B3
ND
1-E
ND
1-E
27U
nkn.
Pro
t.,(A
ccsn
. #A
AH
1085
7)U
nkn.
Pro
t.,(A
ccsn
. #A
AH
1085
7)Larkin et al, EHP 2003
Ubi
quiti
n-co
njug
atin
gU
biqu
itin-
conj
ugat
ing
enzy
me
9en
zym
e 9
ND
96-C
ND
96-C
ND
107-
BN
D10
7-B
ER-a
lpha
ER-a
lpha
Vtg
1Vt
g 1
Cho
rioge
nin
2C
horio
geni
n 2
Cho
rioge
nin
3C
horio
geni
n 3
Vtg
2Vt
g 2
27 27
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
Quantification
-SHMs exposed to several different doses of EE2
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
Choriogenin 3Choriogenin 2
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
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 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 28
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Transferrin AMBP protein (α-1-microglobulin)
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
0 10 100 10001Concentration
0 10 100 1000 1 Concentration
0 10 100 10001Concentration
0 10 100 10001Concentration
0 10 100 10001Concentration
0 10 100 1000
Quantification
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
(ng/L)(ng/L)
1 Concentration
(ng/L)
0 10 100 10001Concentration
(ng/L)
0 10 100 1000
(ng/L)(ng/L)(ng/L)(ng/L)
Larkin et al, EHP 2003 29
2929
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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3131
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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Largemouth bass model
Laboratory exposure • 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
Field analysis
• Site of investigation
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Estradiol Treated (2.5 mg/kg for 48 hrs)
Controls
Estradiol laboratory exposures
Larkin et al, CBP 2002 34
3434
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 * **
Larkin et al, CBP 2002 35
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
)
Larkin et al, CBP 2002 36
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
Need to define “normal” fingerprint pattern in bass before one can identify atypical gene expression patterns in the field
January April August
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Bass reproductive cycle
Need to define “normal” fingerprint pattern in bass before one can identify atypical gene expression patterns in the field
Study site
January April August
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39 39
4040
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)
40
4141
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*
43
4343
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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Acknowledgements
EcoArray Inc University of Florida Barbara Carter Nancy Denslow, Ph.D.
Kevin Kroll
Fish and Wildlife Tara Sabo, Ph.D.
Conservation Commision, Eustis FL
Jamie Kelso Arianna Poston
Jaleh Khorsandian-Falleh Bill Johnson
Bill Farmerie, Ph.D.Li Liu
Anuj SahniUS EPAMichael Hemmer
Ira Adelman, PhD. Funding: Leroy Folmar Ph.D. SBIR grant #1R43ESA011882-01
SBRP (P42 ES 07375) 48
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Additional information
See www.ecoarray.com
Our website contains links to manuscripts using this technology and other information
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Biosensing with Zebrafish
Elwood Linney, Ph. D.
Molecular Genetics and Microbiology
Duke University Medical Center
Biosensing with Zebrafish
Elwood Linney, Ph. D.
Molecular Genetics and Microbiology
Duke University Medical Center
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 50
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Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 52
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Our changing view of “biosensors”
Use results to design new biosensor transgenics
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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 new biosensor transgenics
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Reporter transgenic miceusing constructed retinoic acidresponsive promoter
RARE -TKpr-β gal
RXR RAR
RA
Subset of retinoic acid receptor activity in reporter transgenic mouse:
Reporter transgenic mice using constructed retinoic acid responsive promoter
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 54
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Size relationships, developmental time, changingin size with development:
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, changing in size with development:
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Mouse and zebrafish homeobox genes:
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
Mouse and zebrafish homeobox genes:
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Parallels in axial development between vertebrate species:
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:
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Syntenic relationships
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
Syntenic relationships between vertebrate genomes --genes inherited as linked clusters during speciation
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Retinoic acid indicator embryos
RARE βgal GFP/YFP
RA
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 embryos
RARE-promoter-βgal/GFP/YFP RXR RAR
RA
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Vitamin A-Retinoid Relationships:
retinol dehydrogenase
retinal dehydrogenase(RALDH)
cytochrome p450RAI(cyp26)
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 A-Retinoid Relationships:
retinol dehydrogenase
retinal dehydrogenase(RALDH)
cytochrome p450RAI(cyp26)
RA synthesis
RA metabolism
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Dual in situ hybridization for RALDH2(purple) and cyp26a1(red-orange):
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
Dual in situ hybridization for RALDH2(purple) and cyp26a1(red-orange):
head
tail
RALDH2 in somites
[cyp26 in growing caudal end]
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Model of some of the retinoid events occurring in the trunk neural tube:Model of some of the retinoid events occurring in the trunk neural tube:
18hpf Embryo
Somites
-Raldh2
- RA
- Cyp26A1
Neural tube tail budRA
RA
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 62
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RALDH inhibitor[our work]
Neckless mutant inRALDH2
RALDH inhibitor Neckless mutant in [our work] RALDH2
Begemann, Schilling, Rauch, Geisler and Ingham untreated
Either chemical inhibition of Raldh’s DEAB in zebrafish with DEAB or an isolated
Raldh2 zebrafish mutant produced phenotypes paralleling the mouse Raldh2 knockout phenotypes:
hindbrain changes forelimb or fin inhibition
normal DEAB treated
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 63
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In situs of zebrafish Raldh2 at different developmental stages[Kari Yacisin]:
Point:
In situs of zebrafish Raldh2 at different developmental stages[Kari Yacisin]:
tailbud stage ~10h 10 somites~14h
14 somites~16h 18 somites~18h Point: apparent turn-off of RALDH2 as neural tube ceases to grow
20 somites~<20h 23h
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 64
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Two mouse KO studies ofcyp26A1 revealed caudal truncationsand occasional exencephaly
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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 of cyp26A1 revealed caudal truncations and occasional exencephaly
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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
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 66
66 66
Developmental changes flanking and including neural tube growthwhich we are analysing with 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24hmicroarray analysis:
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 growth which we are analysing with 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h microarray analysis:
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Progression of microarray development: Progression of microarray 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
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 68
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Genes regulated during segmentation
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
Genes 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 our zebrafish toolbox:Some elements of our zebrafish toolbox:
1) live, fluorescent, transgenic embryos
2) anti-sense morpholino knockdown of gene expression
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 70
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Some transgenic lines we made:
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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:
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
Four transgenic lines for examining nervous system:
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Antisense morpholino approaches use byzebrafish researchers to “knockdown” genes
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
Antisense morpholino approaches use by zebrafish researchers to “knockdown” genes
73
7373
no allele) morpholino we injected into a 1-cellzebrafish embryo--these are 4 day larvae afterhatching--the phenotype is what is seen with realmutants in no-tail
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
no--tailtail (T(T--allele) morpholino we injected into a 1-cell zebrafish embryo--these are 4 day larvae after hatching--the phenotype is what is seen with real mutants in no-tail
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Chlorpyrifos studiesChlorpyrifos studies
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 75
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mice
zebrafish
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
mice have AChE plus a butyrylcholinesterase so mouse KOs in AChE allow animals to at least be born and live ~21 days
zebrafish 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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Chlorpyrifos treating zebrafish embryos--our work, high dose(500ng/ml):
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
Chlorpyrifos treating zebrafish embryos--our work, 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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Acetylcholine esterase activity/chlorpyrifos exposure:
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/chlorpyrifos exposure:
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Collaborative work with E. Levin and E. Chrysanthis:
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. Chrysanthis:
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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Targeting acetylcholine esterase with a morpholino:Targeting acetylcholine esterase with a morpholino:
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
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 81
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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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Preliminary comparison of expression of MO-AChE, 100ng/ml CPF10ng/ml CPF versus control using filter of 2x or above expression:
Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC
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 MO-AChE, 100ng/ml CPF 10ng/ml CPF versus control using filter of 2x or above expression:
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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Future--Biosensors:Future--Biosensors: 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
our zCyp26A1pr transgenics with RA inducible promoter
line 1 with 1 uM RA (18h-22h)
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Research support from:
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: NIEHS Superfund and Toxicogenomics Consortium, NICHD
[email protected] http://glowfish.mc.duke.edu
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