Caudal Fin Regeneration in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 301

Figure 22.2 Larval caudal fin regeneration. Larval fin tissue was amputated at 2dpf and
regenerative growth was observed at 0, 1, 2, and 3 dpa.

Fin regeneration proceeds rapidly until the preamputation fin length is reached, at which
point it switches to an ontogenetic growth mechanism. It is speculated that termination
occurs either by an unknown active termination mechanism or by cessation of
regenerative signaling (Iovine, 2007). In an adult fin model, outgrowth occurs from
2 days post amputation (dpa) until the regenerative event is complete by about 14 dpa (at
28 C). Regeneration occurs more quickly at higher temperatures.

22.5 METHODOLOGY

22.5.1 Adult Fin Amputation

Fish between 4 and 12 months are anesthetized with 0.1% tricaine (3-amino benzoic
acidethylester) adjusted to a pH between 7.0-7.5 for approximately 3min or until gill
movements have sufficiently slowed. Anesthetized animals are placed on an inverted
sterile glass petri dish and visualized under a stereoscope. Using a sterile beveled razor
blade, the caudal fin is partially amputated directly anterior to the fin bifurcation. Fin
regeneration takes approximately 12-14 days at 28 C.

22.5.2 Larval Fin Amputation

Two-day-old embryos aremanually or chemically (pronase) dechorionated or 3-day-old
hatched larvae are anesthetizedwith 0.1%tricaine. Using a transfer pipette, anesthetized
animals are placed on agar plates. Using a microinjection rig mounted with a diamond
surgical blade, larvae are amputated just posterior to the notochord (Fig. 22.3). In
comparison to the approach described here, a recent larval fin regeneration study
describes a method that involves fin amputation anterior to end of the notochord (Rojas-
Munoz et al., 2009). While this renders the procedure less technically challenging, the
regenerative model then includes both notochord and fin regeneration mechanisms,
thereby increasing the complexity of cell types and structures that are replaced.

22.5.3 Pronase Treatment to Dechorionate Embryos

Pronase treatment is used to remove embryonic chorions in order to amputate larval fin
tissue at 3 days post fertilization (dpf). To dechorionate embryos, place 50-800

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