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Evolution and Expression of an Alzheimer’s Disease Associated Gene, sorl1 in Zebrafish
Elizabeth Horst and Dr. Wendy Boehmler
Department of Biology, York College of Pennsylvania
http://www.healthhabits.ca/2008/09/29/is-your-dietgiving-you-alzheimers-disease/
http://news.stanford.edu/news/2007/october17/med-fishsleep-101707.html
INTRODUCTION
• Alzheimer’s Disease (AD) is characterized by the aggregation
of senile plaques in the brain that cause deterioration of
mental functions.
RESULTS
Brain Eye
• Decreased sorl1 expression has been shown to increase
pathogenesis of AD (Rogaeva 2007).
Gut
Heart
sorl1
900 bp
segment
• Genes involved in neurodegeneration may also play a role in
neurodevelopment (Bothwell and Giniger 2000).
• The advantages to characterizing genes in zebrafish are
their rapid development, translucent embryos, large
clutches, and the development of in situ hybridization and
morpholino knockdown techniques.
OBJECTIVES
1) What tissues show sorl1 gene expression in
the adult? (Figure 1)
2) What is the evolutionary relationship
between the zebrafish sorl1 gene and other
organisms’ sorl1 gene? (Table 1)
(Figures 3 & 4)
METHODS
Collect mRNA from several zebrafish organs
use RT-PCR to evaluate where sorl1 is present
3’
zebrafish sorl1 gene
1
5,466
Forward Primer
Clone gene into p-drive vector
In Situ Hybridization
-make antisense RNA probe 3’→ 5’
-Antibody probe binds to embryos
-series of washes
-DIG label on probe
3’ end at M13
Ampicillin Resistance
reverse
T7 promoter
6,938 bp
Reverse Primer
Figure 2. p-Drive vector containing
a 900 bp fragment of the zebrafish
sorl1 gene. Plasmid was linearized
and T7 promoter used to synthesize
antisense RNA probe for whole-mount
in situ hybridization on zebrafish
embryos.
Figure 1. Tissue panel showing RT-PCR
results. The primers used were forward
(AGTGAAGATGATCCCAGATG) and reverse
(CTACGCAATGACCATCGGGAC) primers made for a
900 bp section of the 3’ end of sorl1. This
shows that sorl1 is present in brain, eye, gut,
heart and muscle tissue.
Table 1. Syntenic relationship between human
chromosome 11 and zebrafish chromosome 15
supporting an evolutionary link between humans
and zebrafish.
3) What is the spatio-temporal expression
pattern of sorl1 in zebrafish embryos?
• Using PCR it was determined that sorl1 is
present in the brain, eye, gut, heart, and
muscle tissue of zebrafish.
pDrive
Cloning
Vector
900 bp
Muscle
• Aside from its role in amyloid-ß-peptide plaque recycling,
little is known about the role of sorl1 in the brain.
5’
CONCLUSIONS
5’ end at M13
forward
Gene Name
Zebrafish
Chromosome
Human
Chromosome
sorl1
15
11
mre11a
15
11
spcs2
15
11
mgc10485
15
11
cryab
15
11
acad8
15
11
hsp47
15
11
or13.1
15
11
sesn3
15
11
tyr
15
11
clsc
15
11
loc196264
15
11
F
In situ hybridization
M
H
• Using bioinformatics, sorl1 was located on
chromosome 15 of zebrafish and 11 in
humans. These two chromosomes share many
similar genes demonstrating synteny.
• In situ shows where sorl1 is being expressed
spatially and temporally in development.
FUTURE EXPERIMENT
• Determine the neurodevelopmental function of sorl1
using morpholino knockdown of sorl1. Insights into the
functional role of sorl1 may lead to development of
therapeutics for neurodegenerative disorders.
M
MH
E
H
REFERENCES
F
Bothwell, M. and E. Giniger. 2000. Alzheimer’s disease: neurodevelopment converges
with neurodegeneration. Cell 102:271-273.
T
Y
Y
T
Figure 3. Lateral view of
24 hour embryo. Note the
staining in the forebrain,
midbrain and hindbrain
regions. There is an absence
of staining in the yolk or tail.
F-forebrain, M-midbrain,
H-hindbrain, Y-yolk, T-tail
Figure 4. Lateral view
of a 48 hour embryo.
Note the darker staining in
the midbrain-hindbrain
boundary as well as in the
spinal cord. Also notice the
absence of staining in the
forebrain, midbrain or yolk.
E-eye, F-forebrain, M-midbrain,
H-hindbrain, Y-yolk, T-tail,
MH-midbrain-hindbrain barrier
Rogaeva, E., Meng, Y., Lee, J. H., Gu, Y., Kawarai, T., Zou, F., Katayama, T., Baldwin, C.
T., Cheng, R., Hasegawa, H., Chen, F., Shibata, N., Lunetta, K. L., Pardossi-Piquard, R.,
Bohm, C., Wakutani, Y., Cupples, L. A., Cuenco, K. T., Green, R. C. and L. Pinessi.
2007. The neuronal sortilin-related receptor sorl1 is genetically associated with
Alzheimer disease. Nature Genetics 39:168-177.
Woods, I. G., Wilson, C., Friedlander, B., Chang, P., Reyes, D. K., Nix, R., Kelly, P. D.,
Chu, F., Postlethwait, J. H., and W. S. Talbot. 2005. The zebrafish gene map defines
ancestral vertebrate chromosomes. Genome Research 15:1307-1314. Available from:
Google scholar. Accessed 2009 March 23.
ACKNOWLEDGEMENTS
I thank the Pennsylvania Academy of Science for their generous
research grant that is funding this ongoing research. Special thanks to
Dr. Wendy Boehmler for her advice and support in this research.