Transforming Growth Factor-Beta/Activin and Zebrafish Fin Regeneration: Induction of

Cell Proliferation?Shiqiu Li

Dr. Scott StewartStankunas LabUO SPUR 2012

Outline1. Background2. Question/Hypothesis3. Methods4. Earlier Data5. Results6. Conclusion/Finale

Regeneration?Many mammals, like humans, cannot

regenerate lost limbsZebrafish can

What is the mechanism?

What initiates osteoblast differentiation and dedifferentiation?

WNT and BMP!

A Model?

What pathway is involved here?

Transforming growth factor beta/Activin?

ActivinorTGF-β

II ISmad

2P P

• TGF-β and activin are ligands for pathway• Both are part of TGF-β superfamily

• Both have a Type II and a Type I receptor

• TGF-β Type I receptor: Alk5

• Activin Type I receptor: Alk4

ActivinorTGF-β

II I

Smad4

Smad2 P P

ActivinorTGF-β

II I

Smad4

Smad2 P P

Transcription Regulation

Outline1. Background2. Question/Hypothesis3. Methods4. Earlier Data5. Results6. Conclusion/Finale

Question•What role do TGF-β/activin play in zebrafish fin regeneration?

Hypothesis•TGF-β/Activin signaling pathway is involved in cell proliferation in zebrafish fin regeneration

Early differentiation?

Proliferation?

Outline1. Background2. Question/Hypothesis3. Methods4. Earlier Data5. Results6. Conclusion/Finale

Methods

Fixate and Dehydrate

Xylene and paraffin

Section

Mount fin(s)

Fluorescent microscopy

Antibody staining

Collect fish Amputate fish Wait

Alk Inhibitor

X

Amputate fish Harvest fin(s)Short wait

Outline1. Background2. Question/Hypothesis3. Methods4. Earlier Data5. Results6. Conclusion/Finale

Alk4 inhibitors cause reduced proliferation in osteoblasts

Sp7PCNA

Sp7PCNA

Alk4iDMSO Sp7PCNA

Alk4 inhibitors cause reduced proliferation in osteoblasts

× 100

P value = 0.0049

So does TGF-β/activin induce cell proliferation of all cells in the blastema?

Outline1. Background2. Question/Hypothesis3. Methods4. Earlier Data5. Results6. Conclusion/Finale

TGF-β/activin do not induce cell proliferation in the blastema of regenerating fin

TGF-β/activin do not induce cell proliferation in the blastema of regenerating fin

Alk4 inhibitors cause reduced proliferation in osteoblasts × 100

P value = 0.9941

Outline1. Background2. Question/Hypothesis3. Methods4. Earlier Data5. Results6. Conclusion/Finale

Conclusion

• The proliferation of cells in the blastema are not dependent on TGF-β/activin signaling.

• Osteoblast proliferation specifically may or may not be dependent on TGF-β/activin signaling due to differences of Alk4/5 inhibitor doses.

Future DirectionsCo-stain control and Alk4/5 inhibited fins with

osteoblast markers such as Sp7 and Runx2 to fully determine TGF-β/activin’s role in osteoblast proliferation and early redifferentiation.

Inhibit zebrafish with fibroblast growth factor

receptor inhibitor and observe what happens using immunofuoresence.

AcknowledgementsPeter O’DayAdam UngerUO SPURNIHStankunas Lab

Dr. Scott StewartDr. Kryn Stankunas

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