Screening for Drugs that Modulate

1
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/da
nio_rerio.jpg
2
Background Regenerative Medicine in Myths
www.hensleygallery.com
Prometheus
http//www.exoticindiaart.com/artimages/herakles_s
m.jpg
Hercules
3
Observations of Regeneration in Animals
4
Regenerative Medicine
Direct
Indirect
Alternative Animal Model With Regenerative
Capacity
Stem Cell Research

• Relevance
• 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.

5
Research Model- Zebrafish Caudal Fin Regeneration
Outgrowth
Epithelial
Blastema
Wound Cap
Amputation
Cellular
Differentiation
Proliferation
Migration
6
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.
7
Chemical Genetics to Identify Essential
Molecular Targets During Regeneration.
8
Primary Screening Schematic Diagram
9
SB 431542 Inhibits Caudal Fin Regeneration
10
Classifying the Chemical Hits
Biomarkers for specific regenerative stages
11
Secondary Screening - in situ hybridization
mRNA

cDNA
Insert into TOPO vector
PCR to amplify gene
transform bacteria
Transcription labeling
Grow bacteria (amplify)
Linearize DNA
Isolate plasmid
Apply to fixed animal tissue
Detect expression pattern
labeled ss antisenseprobe
www.zf-models.org/images/zf_24hpf.jpg
12
Secondary Screening Group the chemicals to
different regeneration stages In situ
hybridization Marker analysis
13
Results of Secondary Screen
SB431542
No chemical
No chemical
SB431542
dlx5a
dlx5a
raldh2
raldh2
wnt10a
wnt10a
msxe
msxe
14
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.

15
Acknowledgements
Dr. Robert Tanguay Jane LaDu Sumitra
Sengupta Lijoy K. Mathew Dr. Kevin
Ahern HHMI URISC OSU