1. Neural Dependence of Caudal Fin Regeneration
in Zebrafish (Danio rerio)
Sarah Holmes, Airel Estrella, Ankrish Milne, & Nancy Kleckner, Bates College
Introduction
• While most vertebrates have some regenerative capacity, few are
able to regenerate entire limbs.
• Zebrafish (Danio rerio) have the ability to regenerate limbs and
several organs, including fins and heart tissue, after injury or
amputation.
• Because of their readily available genome and rapid regenerative
capacity, zebrafish is a model organism to study regeneration.
• Previous studies have found that fin regrowth is dependent on
the presence of nerves.1
• Although the molecular basis of this nerve dependence is not
well understood, one protein of interest is Agr2+, an anterior
gradient protein that is a homolog of nAg, which is released by
Schwann cells in salamander limb regeneration.2
• The purpose of this study was to observe the effects of AG1478,
a Schwann cell inhibitor, on regrowth, number of axons, and
number of Agr2+ gland cells of the amputated zebrafish caudal
fin.
Methods
• The caudal fins of adult Nacre zebrafish were amputated,
leaving approximately half the fin.
• The fish were incubated for 11 hrs/day for 5 days in either:
• control: 1% DMSO
• treatment: 4 µM AG1478, 1% DMSO.
Data Analysis:
• Stereoscope images were obtained each day post amputation
(dpa) and Photoshop was used to measure fin regrowth.
• Immunohistochemistry and confocal analysis were used to count
axons and Agr2+ gland cells.
Results
References
1Simoes, M. G., Bensimon-Brito, A., Fonseca, M., Farinho, A., Valerio, F., Sousa, S., . . .
Jacinto, A. (2014). Denervation impairs regeneration of amputated zebrafish fins. BMC
Developmental Biology, 14(49). doi:10.1186/s12861-014-0049-2
2Kumar, A., Godwin, J. W., Gates, P. B., Garza-Garcia, A. A., & Brockes, J. P. (2007,
November 2). Molecular Basis for the Nerve Dependence of Limb Regeneration in an Adult
Vertebrate. Science, 318, 772-776. doi:10.1126/science.1147710
Thank you to Nancy Kleckner for mentoring us.
Thank you to MDIBL for this opportunity and to Maine INBRE for
funding this project.
Conclusion
• AG1478 significantly reduced overall caudal fin regrowth
over the course of the 5 days post amputation (p < 0.001).
• AG1478 did not effect the number of Agr2+ gland cells
present in the fin.
• Despite having decreased fin regrowth, the AG1478
treated fish tended to have more peripheral axons than the
controls in the new tissue (p = 0.06).
• These results suggest that fin regeneration is not
dependent on the number of peripheral axons, but rather a
different neural factor.
References & Acknowledgements
Caudal Fin Regrowth
Axon and Agr2+ Counts
Stereoscope image, 1dpa
Acetylated Tubulin/Agr2+Gland Cells
Figure 1. Caudal fin regrowth over 5 dpa. A.) Stereoscope images of
caudal fins. B.) Average caudal fin regeneration of control and
AG1478 fish at each dpa. Control fish exhibited greater regrowth
than AG1478 fish. p < 0.001, two-way ANOVA, mean ± SD shown.
A
B
Figure 2. Average axon count at 5 dpa in regenerated (distal) and
original (proximal) fin tissue per 2500 µm2 box. AG1478 n = 9,
control n = 10. AG1478 fish had more axons in distal tissue p = 0.06,
t-test. No difference in proximal tissue axon count p = 0.86, t-test.
Mean ± SD shown.
Figure 3. Average Agr2+ gland cell count 5 dpa in distal and
proximal fin tissue per 2500 µm2 box. AG1478 n = 5, control n = 5.
No difference between control and AG1478 Agr2+ gland cell counts
in distal, p = 0.91, or proximal, p = 0.36, t-test. Mean ± SD shown.
Future Work
• Successfully stain for myelin basic protein as a marker of
Schwann cells.
– Observe how AG1478 affects proliferation and/or
migration of Schwann cells during regeneration.
– Observe location of Schwann cells compared to
axons in regenerated tissue.
• Confirm that AG1478 specifically inhibits the ErbB
receptors that are required for the development of Schwann
cells.