Transcript Screening for Drugs that Modulate Tissue Regeneration

Screening for Drugs that Modulate
Tissue Regeneration
Jessica Perry
Biochemistry and Biophysics
Dr. Robert Tanguay
Environmental and Molecular Toxicology
6 October 2007
http://www.watergardenersinternational.org/fish/danio_rerio.jpg
Background
Regenerative Medicine in Myths
www.hensleygallery.com
Prometheus
http://www.exoticindiaart.com/artimages/herakles_sm.jpg
Hercules
Observations of Regeneration in Animals
http://www.bergen.edu/faculty/rdill/Biology_II/Bio_II_lab_Images/planaria.jpg
http://pond1.gladstonefamily.net:8080/hydra.jpg
http://www.watergardenersinternational.org/fish/danio_rerio.jpg
http://www.anfibitalia.it/gallery/gallery.asp?ID=24
http://en.wikipedia.org/wiki/Image:House_mouse.jpg
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Regenerative Medicine
Direct
Stem Cell Research
Indirect
Alternative Animal Model
With Regenerative Capacity
Relevance
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Future therapies
Further understanding of disease process
Political issues with stem cells
Alternative animal models of regeneration may prove to be a
complement to stem cell research.
Research Model- Zebrafish Caudal Fin Regeneration
Outgrowth
Epithelial
Wound Cap
Amputation
2 dpa
4 dpa
Cellular
Migration
11 dpa
Blastema
Differentiation
0 dpa
Proliferation
3 dpa
Investigating Zebrafish Fin Regeneration
A. Genetic Mutants
Important genes during development could not be identified - Lethality.
B. Chemical Inhibitors
Tight control of chemical exposure
TCDD inhibits caudal fin regeneration.
3 dpa
DMSO
TCDD
Chemical Genetics to Identify Essential Molecular Targets
During Regeneration.
Primary Screening – Schematic Diagram
SB 431542 Inhibits Caudal Fin Regeneration
3dpa
3dpa
Classifying the Chemical “Hits”
Biomarkers for specific regenerative stages
Secondary Screening - in situ hybridization
mRNA
cDNA
Insert into
TOPO
vector
PCR to
amplify
gene
Transcription
& labeling
cDNA in
vector
transform
bacteria
multiple
copies
Linearize
DNA
Isolate
plasmid
Grow
bacteria
(amplify)
Dlx5a 24hpa
Apply to fixed
animal tissue
labeled ss
antisense
probe
www.zf-models.org/images/zf_24hpf.jpg
Detect expression
pattern
Secondary Screening – Group the chemicals to different
regeneration stages
In situ hybridization – Marker analysis
Junbl Uncut
Junbl 24hpa
Raldh2 uncut
Raldh2 24hpa
Results of Secondary Screen
No chemical
SB431542
dlx5a
dlx5a
msxe
msxe
No chemical
raldh2
wnt10a
SB431542
raldh2
wnt10a
Summary
•Used chemical screens to identify small molecules
that inhibit regeneration
•Developed secondary screens to identify steps in
the regeneration network
•Grouped chemical “hits” to specific regeneration
stages.
Acknowledgements
Dr. Robert Tanguay
Jane LaDu
Sumitra Sengupta
Lijoy K. Mathew
Dr. Kevin Ahern
HHMI
URISC
OSU